HIGH MIOISTURE STRESS -FLOODING / WATER LOGGING

Water logging refers to a condition when water is present in excess amount than its
optinum requirement. It creates an anaerobic situation in the thizosphere due to which the
plant experiences the stress (0, deficient stress).

Nature of Water logging Stress

In the water logged soils. water gets filled in the pores of the soil which are

previously occupied by O,. Such soils suffer O, deficiency.

This O,deficiency depresses growth and survival of plants growing in it.
Flood sensitive plants (eg. Tomato, soybean and sunflower) are killed in the water

logged conditions, while the tolerant species (eg. Rice) withstand water logging for a
considerable time. However, continuous submergence of rice for more than 10 days is also
deleterious resulting in death and decay of the plants.
Plant Water Relations in Flooding Stress
causes lower
The flooding often induces stomatal closure mostly in C3 plants. This
because of reduced root
water flow in these plants. This also results in leaf delydration
water flow from the
permeability. Ultimately, wilting of leaves occus due top the restricted
roots to the shoots.
to the transter of toxic
OccuIrence of these changes in leaves, shoots or roots is due
(acetaldehyde / alcohol) produced under anaerobic conditions in the
roots as well
substances
transpiration stream.
as the levels of PGRS transported from the roots to shoots via
Levels of Endogenous PGRs under Flooding Stress
reduced in the roots.
Endogenous levels of PGRs such as GA and cytokinins (CK)are
shoots causing stomatal closure and
This has enhanced levels of ABA and ethylene in the
early onset of senescence respectively.
that of Aminocyclopropane -]
It is also reported that levels of auxinsare reduced and
biosynthesis are increased under flooding
CarboxylicAcid (ACC). precursor for the ethylene
stress.
during high moisture (tlooding)
Important roles played by these endogenous PGRs
following table.
stress are summarized in the

levels of PGRS and their effect on plants

Effect of flooding stress on the endogenous
 7.
Folia

hig

plantsunder water logging

SI. Level of PGR in Effects on
plants
No.
(Swelling of stem base by collapse or
01. Reduced Auxins Causes "Hypertroplhy"
enlargement of cells in cortex)
enlargement and steim elongation
02. Decreased GA Causesreduction in cell of
senescence and reduced rate
03. Decreased CK Results in early on-set of
assimilate partitioning to the sinks
stonnatal closure with consequential decrease in the
04. Increased ABA Cause
photosynthesis. respiration and
rate of gas exchanges during
the guard cells:
transpiration: results in efflux of K+ from
transpiration;
decreases ion transport due to lower rate of
resulting in
decrease the starch formation in the guard cells
stomatal closure
leaves due to
05. Increased Causes "Epinasty" of leaves (uneven growth of
lower side of the
Ethylene more cellelongation on upper side than the
leaf); induces senescence and Hypertrophy in plants.

transpiration stream,
Thus, the O, stress in the roots under flooding produces signals, via
to the leaves affecting stomatal behaviour ultimately.
Mitigation of High Moisture (Water logging) Stress
stagnating water around the root
1. Providing adequate drainage for draining excessive
system.
for arresting apical donminance and
2. Spray of growth retardant of 500 ppm cycocel
thereby promoting growth of laterals
(MOP)
3. Foliar spray of 2% DAP + 1% KCI
doninance and thus promoting growth
4. Nipping terminal buds for arresting apical
productivity
sympodial branches (as in cotton) for increasing
of 40 ppm NAA for controlling excessive pre-mature fall of
5. Spray
flowering buds/young developing fruits and pods
increasing photosyntheticactivity
6. Spray of 0.5 ppm brassinolide for
7. Foliar spray of 100 ppm salicylic acid for increasing stem reserve utilization under
high moisture stress
8. Foliar spray of 0.3 % Boric acid + 0.5 % ZnSO4 + 0.5 % FeSO, + 1.0 % urea during
critical stages of the stress.
 REVIEW ARTICLES

Oxidative stress and antioxidative system in plants

Ajay Arora, R. K. Sairam* and G. C. Srivastava
Division of Plant Physiology, Indian Agricultural Rescarch Institute, New Delhi 110 012, India

Free radicals and othcr active derivatives of oxygen

of active oxygen in plant tissues, having potential to
are inevitable by-products of biological rcdox reac gencrate singlet oxygen, 'O; and superoxide, O;. The
production of actíve oxygen is an unavoidable conse
tions. Reactive 0xygen species inactivate enzymes
and damageinportant cellular componcns. The quence of the operation of the photosynthetic electron
inereased production of toxic oxygen derivatives is ransport chain in an oxygen atmosphere.
considered to be a universal or çommon feature..of. The major. OXYgen-consuming procesSes associated
stress conditions. Plant and other organisms have with photosynthesis are: (a) the oxygenase reaction of
evolved a wide range of mechanisms to contend with ribulose-1,5-bisphosphate carboxylase (Rubisco), which
this problem. The antioxidant defence system of the is the initiating reaction of the photorespiratory path
plant comprises avaricty of antioxidant molecules way, and (b) direct reduction of molecular oxygen by
and enzymes. The effects of the action of free radi the photosystem 1 (PSI) clectron transport chain. In
cals on membranes include the induction of lipid
peroxidation_ and fatty acid de-esterification. Both addition, certain photosystem I (PSII) components are
cthylene biosynthesis and membrane breakdown, also capable of converting molecular 0, to high-energy
which appear to be closcly linked, seem to involve singlet oxygen (O). A cyanide-insensitive respiratory
frec radicals, although the sequence of events gener pathway in chloroplasts that competes for electrons with
ating these free radicals is still poorly understood. 1t photosynthetic electron transport" may also roduce oxy
is clear that the capacity and activity of the antioxi gen.
dative defence system are important in limiting oxi Oxidative stress is essentially a regulated process, the
dative damage and in destroying active oxygen equilibrium between the OxÍdative and. antioxidative.
species that are produced in excess of those normally capacitics determining the fate of the plant. Under non
required for metabolism. Transgenic plants offered stressful conditions the antioxidant defence system pro
us a means by which to achieve complete under vides adequate protection against active OXy gen and
standing of the roles of the enzymes involved in pro ree radicals. Both atural and man-made stress_situa
tection against stress of many types, environmental
and induced. Studies on transformed plants express tions provoke incrcased production of toxic oxygen
ing increased activities of single enzymes of the anti derivatives. In response, thc capacity of he antioxida
oxidative defence systen indicate that it is pos sib le tive defence system is increased.. But in most situations
to confer a degrec of tolerance to stress by these the response is moderate'. Furthemore, some important
means. The advent of plant trans formation has sites such as the reaction_centre protein of PSIl (DI) and
placed within our grasp the possibility of engincering the apoplastic space, appcar o have very little protec
greater stress tolerance in plants by enhancements of tion against oxidative damage8,9
the antioxidative defencce sys tem.

Generation of toxic reactive oxygen species and

LIVING organisms are cxposed o different kinds of associated regulatory mechanisms
stresses, which may originate from human activities or
natural causes such as air pollution, drought, tempera Molecular oxygen is produced as a result of the oxida
ture, light intensities and nutritional limitation. Since tion of water by the photosynthetic electron ransport
plants have limied mechanisms of stress avoidance, chain. The latter, however, can also use oxygen as an
they require flexible mcans of adaptäion to changing clectron acceptor (Figure 1). In addition, molecular oxy
environmental conditions. A common feature of differ gen ASsimilated during photorespiration__.pruducing
ent stress factors is their potential to increase the pro phosphoglycollate. Both of these reactions have positive
duction of reactive oxygen speçis. in plant tissues. and negaiveettects. Superoxide, pruduced by the
Reactive oxygen species are also generaled in plant ransport of clectrons to oxygen, is not compatible _with
cells during nonnal metalolic processes'. The photo metabolis1m and must be eliminated by the antioxidative
synthetic electron transport system is the major source defence system while recycling of phosphoglycollate to
phosphoglycerate (in order to re-enter the Benson
Calvin cycle) results in a considerable loss of assimi
*For correspondence. (e-mail: rks ppl@yahoo.com) lated carbon. In addition, large aanounts of H,O) are

CURRENT SCIENCE, VOL. 82, NO. 10, 25 MAY 2002 1227

 REVIEW ARTICLES

produced during the oxidation of the glycollate in he excited chlorophyll to the ground state. Thermal energy
peroxisomes. Although much of this H,0; is destroyed dissipation plays a pivotal role in photoprotection since
by catalase, some chemical decarboxylation of keto it limits the rate of reduction of the first stable electron
acids by H,0, still occurs'", Nevertheless, photosyn acceptor of PSII (QA).
thesis benefits since photorespiration protects the pho
tosynthetic membrane against light-inducod damage at
times when carbon assimilation is limited' This may Superoxide production
indeed be regarded as the principal function of photo
respiration, which is far more effective than electron Photo-reduction of dioxygen in chloroplasts was first
transport to oxygen (termcd pseudocyclic clectron flow shown by the production of acetaldehyde in the pres
or the Mehler reaction) in protccting against photoin cnce of cthanol anxd catalase and the photo-reduced
hibition14 product was assumed to be hydrogen peroxÍde'". Subse
quently, in the 1970s the primary reduced product was
identified as the superoxice anion radical (0,). Under
Formation of singlet oxyge most circumstances, the control of electron flow be
twccn PSII and PSI regulates the reduction state of the
The chlorophyll pigments associated with the clectron acceptor side of PSI. This means that the redoX state of
transport system are the primary source of singlet oxy PSI acceptors does not significantly limit electron trans
gen ('O; Figure 1). Singlet oxygen may also arise as a port'. The regulated activation of Benson -Calvin cycle
by-product of lipoxygenase activity. Like the hydroxyl and control of the rate of electron flow are important
radical, OH, 'O, is highly destructive, reacting with factors deternining the redox state of the ferredoxin
most biological molecules at near diffusion-controlled pool'",0, This is important because ferredoxin and the
rates!$.16 The lifetime of excited chlorophyll singlet
state is short within these aggregates, but varies accord
electron carriers on the reducing side of PSI have
sufficiently negative electrochemical potentials to
ing to physiological conditions. The excited singlet state donate electrons to oxygen resulting in the fomation
of chlorophyll is used for the transfer of energy or clec of superoxide radical ; (Figure 1). There are two
trons. However., there are two other possible routes of sites of 0, production on the reducing side of PSI'21
de-excitation, radiative decay (fluorescence) and Con The majority of O2 reduction in vivo is thought to pro
version to the triplet chlorophyll state. The latter inter ceed via reduced ferredoxin (Fdred), which reduces
acts with Oxygen to produce'O,. molecular OXYgen the superoxide radical (Reaction
to

There are two strategies for defence against O, in the 1). Hydrogen peroxide is then formed through dismuta
thylakoid membranes. The first is the regulation of the tion of O; (Reaction 2). The latter occurs spontane
light-harvesting apparatus to minimize triplet chloro ously, but the velocity of the reaction is greatly
phyll production, and the second is the rapid quenching increased by SOD (Reaction 3).
of both he triplet chlorophyll state and O, by mem
brane-bound quenchers. Two major processes decrease 202 + 2Fdyed ’ 20, + 2FdoKs (Reaction 1)
the lifetime of excited sínglet-state chlorophyll; the irst
is photochemistry and clectron transport in the reaction 20, + 2H* ’ H02 + O2, (Reaction 2)
centres and the second process involves thermal dissipa
SOD
tion of excess excitation energy that quenches singlet 20, + 2H* H; O, + O2. (Reaction 3)

Production and scavenging of hydrogen peroxide

STROMA NADP in chloroplasts
0,
FdFes
Hydrogen peroxide is produced by the dismutation of
superoxide radicals in a reaction mostly catalysed by
PSI Cy1 B,
PSI, superoxide dismutase (Reaction 3". In leaf cells, ata
lase is exclusively localized in peroxisomes and has not
POH
been found in chloroplasts. The hydrogen peroxide in
H,0 PC PC chloroplasts is scavenged by a peroxidase reaction using
’0, the photo-reductant produced in the thylakoid as the
electron donor". Thus, diffusion of hydrogen perox
THYLAKOD LUMEN ide from chloroplasts to peroxisomes and its scavenging
CHH*
by catalase are very unlikely to occur. The electron
Figure 1. Production of superoxide radical and singlet oxygen in donor for the peroxidase. reaction. has been identified as
ascorbate25
chloroplast at the site of PSI and PSII.
1228 CURRENT SCIENCE, VOL. 82, NO. 10, 2s MAY 2002
 REVIEW ARTICLES

superoxide couple has an Em at pH 7 of around

Catalase
2H,02 2H,0 + O:, -330 mV (ref. 27).)
Peroxisomes

Ascorbatepcroxidase
Hydroxyl radical: The most reactive oxidant in
H,O, + Ascorbic acid cells
Chloroplas
H,0+ Mono-dehydro-ascorbic acid.
Ilydrogen peroxide axd superoxide radical (0;) by
themsclves are relatively less damaging, but they can
form specics damaging the essential cellular compo
nents such as hydroxyl radicals (OH) hat can initiate
Superoxide generation in plant mitochondria lipid peroxidation and also attack DNA, proteins and
many small molecules. Fenton, in the late nineteenth
Several pathways of oxygen consumption are poten
tially available to isolated plant mitochondria or sub
century described the oxidizing potential of bydrogen
peroxide with ferOus salts. Forty years later, Haber and
milochondrial particles26 These can be identified as
follows:
Weiss0 identificd hydroxyl radical (OH)) as the oxidiz
ing species in these reactions.
1. Oxygen consumption via cytochromne oxidase to
for more Fe +H,0, ’ Fe +OH +OH.
produce water., a process which accounts
than 95% oxygen consumption in nomal, cyanide
sensitive mitochondria (Figure 2). In biological systems availability of reduced ferrous ion
may limit the reaction, but feric ion can be recycled to
2. Direct reduction of oxygen to superoxide anions in reduced ferrous state by reducing agents such as 0,.
the flavoprotein region of NADH dehydrogenase
segment of the respiralory chain. The component
Fe + 0; ’ 0, + Fe.
responsible is likely to be the flavoprotcin (of either
intermal or extermal dehydrogenase) or perhaps an Therefore, this reaction can be summarized as:
iron-sulphur centre (Figure 2). The process may be
identified by its insensitivity to KCN, antimycin A
and salicylhydroxamic acid and by the sensitivity of Fe*,Fe
H,O, + 0, OH +0H +Oz.
the assayed epinephrine oxidation rate to superoxide Haber- Weiss reaction
dismutase.
3. Oxygen reduction to Superoxide anions in the Thus in the presence of trace amounts of iron ion,
ubiquinone-cytochrome the respiratory
region of hydrogen peroxide will form the
identified by its insensi superoxide and
chain. The process may be destructive hydroxyl radical, and initiate the oxidaion
tivity to saticylhydroxamic acid and antimycin A, its of organic substrates. Metal ions such as Cu, Qu can
sensitivity to KCN and the sensitivity of the assayed replace Fe, Fe in these reactions.
rate to superoxide dismutase. In these schemes,
ubiquinone donates an electron to
fully-reduced
cytochrome C and lcaves an
unstable, highly Oxidation of organicsubstrates by hydroxyl
semiquinone species, which would nor radical
reducing
mally reduce cytochrome bs66. It is presumably this
unstable semiquinone or a closely interacung species Oxidation of organic substrates may proceed by two
which reduces the oxygen to superoxide anion, since possible reactions: (1) addition of OH to an organic
only a spocies at this sile would have enough reduc molecule, or (2) abstraction of a hydrogen atom fronm it.
In the aklition rcaction he OH add to organic substrate
ing potential for the reaction. (The Oxy gen

1/2 O,

NADH FAD UBIQUINONEyt b oyt o cyte Goyt

H,o

Figure 2. Sites of superoxide radical formation in mitochondrial electron transfer

system.

1229
CURRENT SCIENCE, VOL. 82, NO. 10, 25 MAY 2002
REVIEW ARTICLES k n o w n

stress
d a t i v e

forming a hydroxylated product, which is further Conditions enhancing oxidative calle

oxidized by Fe* ion, O or other agents to stable oxi increased under

The generation of toxic O, species is
the

dized product. The hydroxylated product can also dis
mutate to fom cross-linked products.
R+ OH ’ ROH,
ROH' + Fe ROH +Fe,
ROH + 0, ’ ROH +0;,
ROH + ROH + 2H ’ R- R+ 2H,0.
In the abstraction reaction, the OH radical oxidizes the
organic substrate by forming water and an organic radi
cal. The latter product has single unpaired clectron and
thus can react with oxygen in triplet ground state. The
addition of triplet oxygen to the organic radical can lead
to the formation of a peroxy-radical, which can readily
abstract hydrogen from another organic molecule lead
ing to the formation of a second organic radical. This
chain reaction is far more damaging than any other
reaction catalysed by reactive oxygen species.
RH + OH ’ R+ H,0,
R +O ’ ROO,
ROO +RH ’ R+ROOH.
This hydrogen albstraction reaction of hydroxyl radical
is best demonstrated by lipid peroxidation of linolenic
acid in cell membranes (Figure 3).
The lipid peroxides (ROOH) are unstable in the pres
ence of Fe or other reduced mnetal ions (such as Cu),
as they participate in a Fenton reaction leading to the
formation of reactive alkoxy radical.
ROOH + Fe ’ OH + Fe 4+ RO,
Thís alkoxy radical is as damaging as the hydroxyl radi
cal, thus starting a cascade of oxidative reactions.
H: H:0:H
Degradatáon
Products
conditions, Plants which are exposed to severe
stress
susceptibility to
stress, have been shown to increase chlo
photo-inhibition with subscquent development of
tOsig3 Photo-oxidative damage is exacerbated by
atmospheric pollutants, herbicides, hcavy metals
and
cercosporin (which originates
natural compounds like
from fungi of the genus Cercospora).
Pollutants
Atmospheric pollutants such as ozone (O3) and sulphur
dioxide (SO;) have been implicated in free-radical for
Imation34,3s and are considered to be one of the major
factors influencing modern forest decline. Ozone, which
originates rom a natural photochemical degradation of
nitrous oxide (NO,), seems to be a greater threat to
plants than SO, (ref. 36). Mehlhorn* suggested that the
phyto-toxicity of O3 is due to its oxidizing potential and
the consequent formation of radicals that induce free
radical chain reactions. The O, concentration in the
intercellular air spaces of leaves is close to zero. Thus
ozone is unlikely to reach the chloroplast, but it never
theless causes pigment bleaching and lipid peroxida
tion. Stimulation of synthesis and degradation of the
PSII-DI protein occurs in spruce trees following O,
treatment" and a decrease in the activity and quantity
of Rubisco has been found in poplar following exposure
to O3 (ref. 40).
Exposure to SO results in tissue damage and release
of stress ethylene from both photosynthetic and non
photosynthetic tissues. Fumigation with SO, causes a
shift in cytoplasmic pH. The proton concentration of the
cytoplasm is one of the most important factors regulat
ing cellular activity. When cells are exposed to SO an
appreciable acidification of the cytoplasm Occurs,
because this gas reacts with water to form sulphurous
acid which may then be converted into sulphuric
acic2,. The oxidation of sulphite to sulphate in the
chloroplast also gives rise to the fomation of O, (ref.
44). The oxidation of sulphite is initiated by light and is
mediated by photosynthetic electron transport. This
results in loss of photosynthetic function caused by
inhibition of the activity of SH-containing, light
activated enzymes of the chlorplasS,t6.
Herbicides

Several herbicides have becn found to generate active

Oxygen species, either by direct involvement in radical
production or by inhibition of biosynthetic pathways.
Figure 3. Hydroxyl radical-mediated peroxidation of linolenic acid The bipyridinium herbicides generate Oxygen radicals
in lipids. directly in light. Compounds such as paraquat (also
1230 CURRENT SCIENCE, VOL. 82, NO. 10,25 MAY 2002
 REVIEW ARTICLES

known as methyl viologen) induce light-dependent ox0 tions, including chilling, high light, drought and

dative damage in plants. Members of this group are paraquat, oxidative stress ensures primarily due to

called total-kill' herbicides". The di-cationic nature of the decrease in antioxidant defences but also due to the
these compounds facilitates their reduction to radical increase in free-radical production mediated by catalytic
cation. The PSI-mediated reduction of the paraquat Fe.
di-cation results in the formation of a mono-cation radi
cal, which then reacts with molccular oxygen to pro Photosensitizing toxins
duce O; with the subsequent production of other toxic
species, such as H;0, and OH (ref. 32). Natural photosensitizers induce oxidative damage in the
The diphenyl ethers, cyclic imides and lutidine
light, and make plants sensitive to visible wavelengths
derivatives act by inhibition of biosynthetic pathways of light and cause phytotoxic reactions. Perhaps the
with the subsequent accumulation of reactive, radical best-known fungal photosensitizer is cercosporin. The
forming intermediates. The mode of action of these her fungus Cercospora causes worldwide destruction of
bicides is based on their ability to induce the abnomal
important crop spocics, including Com, sugar bect,
accumulation of photoscnsitizing tetrapyroles, specifi tobacco, coffee, soybean, and banana. Cercosporin is a
cally protoporphyrin°. It is somewhat anomalous that red, perylenequinone secondary metabolite produced by
the reaction product protoporphyrin IX, accumulates in many species within the genus. When activated by light,
conditions where the enzyme which catalyses its forma it reacts with oxygen to form '0, (ref. 56). lon leakage
tion is expected to be inhibited. rapidly results due to changes in membrane composition
Other compounds such as diuron, that block photo caused by lipid peroxidation" The membrane damage
inhibitors of
caused by cercosporin provides nutrients for fungal
and caro
synthetic electron transport
tenoids biosynthesis such as norflurazon, initiate growth and sporulation in the host.
photo-oxidative processes most probably via he genera
tion of 'O, (ref. 15). Herbicides which block photosyn
thesis cause increased excitation energy transfer from Drought
triplet chlorophyll to oxygen, while those which inhibit
carotenoids biosynthesis eliminate important quenchers A plant's response to drought stress is a complex phe
ofthe triplet chlorophyll and 'O. nomenon that appears to involve the synthesis of poly
amines and a new set of proteins whose function
is largely unkown8 Abscisic acid is central in the

Metals response because it stimulates stomatal guard cells to

close, reducing water loss. This process also reduces the
Accumulation of phytotoxic metals results from indus availabiliy of CO2 for photosynthesis, which can lead
trial and agricultural practices. Zn, Cu and Cd are widc to the formation of rcactive oxygen species from the
misdirecting of clectrons in the photosystem. Hence,
spread pollutants resulting in stunted growth, chlorosis mechanisms that reduce oxidative stress may play an
and necrosis"*.
Copper (Cu) ions cause light-mediated
important role in drought tolerance.
lipid peroxidation, pigment bleaching rye
Cu
and
ions cytosolic CWZn-SOD Was induced
decline in endogenous catalase (CAT) level. tomato,
are redox active and catalyse Fenton-type reactions pro strongly by drought, while the chloroplastic CuZn-SOD
remained largely unaffected. Glutathione reductase acti
ducing OH (ref. 32). Lipid peroxides also originate
from the induction of lipoxygenase in he presence of vity increased in drought-stressed wheat and cotton
plants, and it was propOscd that, in addition to remov
Cu. This enzyme is known to initiate lipid peroxida
tion. Cadmium treatment decreases chlorophyll and ing HO2, this incrcase might make NADP available
heme levels of geminating mungbean seodlings by that can accept electrons from ferredoxin, thereby
minimizing chances of superoxide fomation. In
induction of lipOxygenase with the simultaneous inhibi drought-tolerant Hordeuwn species, levels of glutathione
tion of the antioxidative enzymes, SOD and CAT.
Such inhibition results from binding of the metal to the rvductase and ascorbate peroxicase increased, but SOD
important sulphydryl group of enzymes, which increase activity was not examinedÑ0 Drought-stressed cotton
the phytotoxic action of metals. Iron has a pivotal and was found to be resistant to a subsequent challenge of
paraquat, which may indicate the existence of a com
59
dual role in free-adical chemistry in all organisms. Free mon proective mechanism against these stresses.
Fe can participate in Fenton reactions and catalyse the in lipid peroxication and
generation of hydroxyl radical and other toxic oxygen Drought-induced changes
the activities of SOD and catalase were compared in
species. On the other hand, Fe is a constituent of the
antioxidant enzymes catalase, ascorbate peroxidase, two mosses, the drought-tolerant Tortula rralis and
guaiacol-peroxidase and ferro-superoxide dismutase. the drought-sensitive Cratoneuron flicinun". During
When plants are exposed to a variety of adverse condi stress, the drought-tolerant moss showed lower levels of
1231
CURRENT SCIENCE, VOL. 82, NO. 10, 25 MAY 2002
 REVIEW ARTICLES

lipid peroxidation together with increased levels of both brane integrity) has been put forward. Initally, a trans
the enzymes: the opposite occurred in the sensitive formation of the lipids leads to membrane breakdown.
moss. Glutathione metabolism was subsequently studied Free radicals are then produced by peroxidaion, arnd
in the tolerant moss, and oxidized glutathione (GSSG)
these free radicals promote the burst of ethylene. The
was found to be a good indicator of drought stress°
effect of the rise in ethylene is, therefore, to accelerate
Drought-tolerant and intolerant maize inbrcd werc ana the sencscence. This hypothesis is in agrecment with the
lysed by Malan et al." and resistance was found to cor 73
relate with uZn SOD
work of Mayak and Adam", who suggested that cthyl
and glutathione reductase ene synthesis requires mcmbrane deterioration so that
activities, although higher levels of one enzyme alone ACC, a polar molecule, may approach the ethylene
apparently did not confer drought tolerance. forming cnzyme, the membrane enzyme that transforms
Sairam et alb,os showed that H,0, scavenging sys ACC into cthylene.
tems represented by ascorbate peroxidase, glu
Aerobic respiration, which is strongly inhíbited by
tathione reductase and catalase are more important in
cyanide (CN) and azide (N;) ions, is found to contiue
imparting tolerance against drought-induced oxidative even when cytochrome oxidase is blocked by these
stress than superoxide dismutase alone. The relative
inhibitors in certain organs andor tissues, due to the
tolerance of a genotype to water stress as reflected by existence of an altemate short branch in the electron
its comparatively lower lipid peoxidation and higher transport pathway at the first step involving ubiquinone.
membrane stability index, chlorophyll and carotenoid This provides a means for continued oxidation of NADH
contents was closely associated with its antioxidant
and operation of the TCA cycle. The alternate pathway is
enzyme system (SOD, APO, GR, CAT). highly significant in the respiratory climacteric of ripen
ing fruit and leads to the production of hydrogen perox
Free radicals in senescence and ripening ide and superoxide, which in tum enhance the oxidation
and breakdown of memnbrane, necessary activities in the
14
ripening process. Solomos' pointed out that ethylene
Membrane brcakdown and ethylene biosynthesis, which may act to implement the alternate pathway in ipening
appear to be closely linked, seem to involve free radi fruits. In tomato it was observed that cyanide-resistant
calso6 In vitro studies have suggested that the conver respiration constitutes about 94% of the total respiration
Sion of ACC to ethylene may involve peroxidation
reaction67 Studies performed with Dianthus caryophyl
in ripe fruits, whereas in unripe fruits it is about 60% of
the total respiration'. In certain cases, its involvement
lus indicate that senescence can be slowed by retarding has been noted in raising the temperature as in ripening
peroxidation by neutralizing free radicals. Moreover, banana76 and ipening mango". Cyanide-resistant respi
inhibition of ethylene bursts slows peroxidation and ration increases during ripening of tomato fruits along
prolongs the life of cut camations, suggesting a rela with the activity of hydrolysing enzymes, suggesting
tionship between free radical generation and cthylene that both the processes are related
productionó8 Free radicals and antioxidants also play a significant
Sylvestre et al. showed that during petal development role during the natural senescence process. Dagmar et
in cut carnation, ehylene content increases simultane al. have reported in the case of maize that early senes
ously with peroxidation and the activities of SOD and cence of cv. X 3342 was due to the enhanced HO, pro
catalase decrease from the initial stage to blooming, duction and lipid peroxidation, and lower SOD,
MCRae et al." demonstrated moe precisely the role of especially Mn-SOD, AsAPOD, and CAT activity
superoxide anion in this reaction. Baker et al" reported towards maturity compared to the late maturing cv.
that the vase-life of camations was increased by the use of Deccan 103.
sodium benzoate (a free-radical scavenger) at a concentra
tion of 10 M, and that the outburst of ethylene was inhi
bited. According to Mayak et al"", the microsomal mem Antioxidative system of plants
branes of carnation petals produce an increasing amount Plants possess very efficient scavenging systems for
of O, radicals during scnescence, and this increase paral
lels the decrease in membrane fluidity. The addition of a reactive oxygen species that protect them from destruc
tive oxidative reactions. These defences are not
free-radical scavenger, propyl gallate al a concentration of restricted to the intracellular compartment, but are also
10 M, prevents change in he fluidity of the microsomal found in he apoplast to a limited extent.
membranes. The O, anions contribute to brcakdown of
phospholipids and the fatty acids releascd may then be
peroxidized". This phenomenon leads to he rigidification Superoxide dismutase
of the membrane in senescent tissues.
The following hypothesis concerming the sequence of Superoxide dismutase, the family of metalo-enzymes,
cvents (and in particular those believed to affect mem catalyses the disproportionation of superoxide O, to
1232 CURRENT SCIENCE, VOL. 82, NO. 10, 25 MAY 2002
 REVIEW ARTICLES

molecular OXygen and Hz0, (ref. 80). Superoxide dis immediately to Hz0, to be98-100
scavenged by he membrane
mutase removes superoxide and hence decreases the bound ascorbate peroxidase Isolated intact chloro
risk of hydroxyl radical formation from superoxide via plasts rapidly metabolize exogenously added Hz0, (refs
the metal-catalysed Haber-Weiss-type reaction. Three 98-100), indicating that in siu the chloroplasts may
isozymes of SOD, namely Mn-SOD, Cu/Zn-SOD climinate H,0, generated both intermally and extemally.
and Fe-SOD have been reported in various plant spe Two enzymes are involved in the regencration of redu
cies. Mn-SOD is predominantly found in mitochon cod ascorbate, namely mono-dehydro -ascorbate reduc
dria81,82 and peroxisomes83-85 However, there are also tase (E.C. 1.6.5.4) which uses NAD(P)H directly to
reports of its occurrence in the soluble cytosolic frac recycle ascorbate (Figure 4) and dehydro-ascorbate
tion86,87 reductase. However, the situation is further complicated
CWZn-SOD initially supposed to be located
the cytosolic fraction2,87,88 has lately also been because mono-dehydro-ascorbate itself is an efficient
in
chloroplastic2,88,89 and mitochondrial electron acceptor101,102 Mono-dehydro-ascorbate is red
reported from
fractions88,90 Similarly, Fe-SOD though predominantly uced directly to ascorbate using electrons derived from
the photosyntheticelectron transport chain as follows:
detected in chloroplasts, has also been reported from
cytosolic®6 mitochondnai91,92 and peroxisomal? firac
tions. It will thus seem that almost all of the isozymes Light
of SOD bave been detected in most of the cellular com 4 Mono-dehydro -ascorbate + 2H,0.
Electrontransport
ponents. 4 Ascorbate + O.

Ascorbic acid and ascorbate peroxidase The regencration of ascorbate within the chloroplast
provides a putative mechanism for the regulation of
Ascorbate is present in chloroplasts, cytosol, vacuole electron transpor. Ascorbate is not only a potent anti
and apoplastic space of leaf cells in high concentra oxidant, but is implicated in the pH-mediated modula
tions93,94 tion of PSII activity and its down-regulation associated
It is perhaps the most important antioxidant in
with zeaxanthin formation", This is a potent mecha
plants, with a fundarmental role in the removal of hydro
gen peroxide99 Oxidation of ascorbate Occurs in two nism for photo-oxidation. The Mahler
preventing
sequential steps, first producing mono-dehydro peroxidase reaction sequence helps to generate the low
ascorbate, and if not rapidly re-reduced to ascorbate, the lumen pH values required for the formation of zeaxan
mono-dehydro-ascorbate disproportionates to ascorbate thin 104,10s This xanthophyll pigment has been consis
be involved in the mechanisms of
and dehydro-ascorbate (Figure 4). tently shown to
106
Ascorbate activity has mainly
peroxidase been thermal energy dissipation'
reported from chloroplast and cytosol°. However Some

recent Studies have also reported its occurrence in mito

Glutathione and glutathione reductase
chondria as welß9,97, In the chloroplasts, SOD and
ascorbate peroxidase enzymes exist in both soluble
and thylakoid-bound forms. Superoxide generated at the
Glutathione, glutamyl cysteinyl glycine (GSH) is the
membrane surface Can thus be trapped and converted major low molecular weight thiol compound in most

2H*
SOD Non-enzymatic
reaction
Glutathione AADPH
NADP
H0: oxidized
Acid

Mopdehydro Dehyf-ascorbate Gluti thione

Asprbate Redu tase
Perqxidase Ascorbe reductase Reductase
NADPII
NADP
H,04 Monodehydro DehydrQ Glutathione
Ascorbute reduced
Ascorbate Non-enzymatlc
reaction

scavenging and ascorbic acid regeneration involv

Figure 4. Asada--Halliwell pathway of hydrogen peroxide
ing various antioxidant enzymes.
1233
2002
CURRENT SCIENCE, VOL. 82, NO. 10, 25 MAY
 REVIEW ARTICLES
plants 07, 108
Glutathione acts as disulphide reductant to detectable Mn-SOD or Fe-SOD activity, respectively'
affected
minimal medium, aerobic growth was
protect thiol groups on enzymes, regenerate ascorbate
exacer
and react with singlet oxygen and hydroxyl radicals. In by methyl viologen, a bipyridly herbicide that Mn
Some plants, such as legumes, homoglutathione (gluta bates O; production. The Sod A mutant, lacking
sensitive methyl
myl cysteinyl alanine) may partly or wholly replace SOD activity, Was cspecially to
glutathionel05 It acts as a protein disulphide reductant, treatment, although neither single mutation
viologen
which detoxifies herbicides by conjugation, cither spon affected acrobic cell growth. Sod A-Sod B double mu
taneously or by the activity of one of a number of glu tants were unable to grow on minimal medium under
tathione-S-transferases,
in
and regulates gene expression aerobic conditions. These double mutants demonstrate
attackresponse how harmful superoxide anion formation is for bacterial
to
environmental stress and pathogen
l07, 110-112
. It also participates in the regencration cells. In addition, the double mutant was killed by expo
of ascorbate from
dehydro-ascortbate via the enzyme
dehy dro-ascorbate reductase sure to H,0, and had a much higher spontaneous muta
(E.C. 7.8.5. 1; Figure 4). In tion frequency in the presence of oxygen than wild type
such reactions GSH is oxidized to
phide (GSSG). GSH is glutathionc disul cells with normal SOD activity l6.
regenerated by glutathione In Contrast to the harmful effect of the double
reductasc (GR) in a
4). Creissen e al. have NADPH-dependent rcaction (Figure
reported that GR is located in
mutant, reports of the consequences of increased SOD
activities by complementation in null mutants and SOD
chloroplastic, mitochondrial and cytosolic fractions of Over expression are very inconsistent. Overproduction
maize. Recent studies have further
tence confirmed its exis
in chloroplast, mitochondrial and cytosolic frac of plasmid encoding Mn-SOD led to increased sensitiv
tions87,89 ity of E. coli cells to methyl viologen"" Increased
Hz0: production might be responsible for this observed
increase in sensitivity. Hopkins et al. have reported
Dehydro-ascorbate+ 2 GSH DHAR
Ascorbate that Mn-SOD and Fe-SOD
functionally
are not
+GSSG, equivalent and that they have different antioxidant roles
in E. coli. Mn-SOD was more effective than Fe
GSSG + NADPH GR
’ 2GSH + NADP. SOD in preventing DNA damage, while Fe-SOD
excelled in protecting a
cytoplasmic superoxide-sen
a-Tocopherol and carotenoids sitive enzyme.
Only one form of GR has been identified in E. coli.
The membrane-associated antioxidant, Both the GR enzyme proteins and the gor gene from E.
Ctocopherol,
scavenges singlet OXYgen and lipid peroxides. Ascor coli have been characterized in detaij 19 Barbado et
bate and ocopherol are extremely effective al l20 demonstrated that a bacterial mutant deficient in
antioxi GR activity had increased sensitivity to H,0 relative to
dants because they are relatively poor electron
donors
in physiological conditions and act primarily by
transfer
a catalase-deficient parental strain. The GR-efficient
of single hydrogen atoms'4 Ascorbic acid and Ctoco mutant expressing the gor gene had a greatBy increased
pherol can scavenge hydroxyl radicals, singlet Oxygen GR activity, which was directly elated to an increased
and superoxide radical. glutathione content. However, overproduction of GR
could not replace SOD in a SOD-deficient mutant, and
Carotenoids quench singlet oxygen and also protect GR was less effective than
by absorbing excess excitation energy from chlorophyl Fe-SOD in protecting
by direct transferl03- 106 against methyl viologen toxicity21.
Catalase prevents oxidative damage to DNA during
aerobic growth. E. coli produces two distinct foms of
Protection against oxygen radicals - The catalase, a bifunctional
catalase-peroxidase
molecular approach oxidase I, HP-I) and a monofunctional catalase(hydroper
(HP-I).
However, studies on the physiological role of these
The availability of null mutants in bacteria allows rela
catalases Oxidative stress are complicated because
interest. the two catalasesS are genetically controlled by three
tively simple complementation with genes of loci: kat E kat F and kat G (ref. 122). The genes kat G
Therefore, our current knowledge about the physio
antioxidative enzymes has been and Kat E encode HP I and HP II, espectively and
logical function of obtained in well do not appear to be linked. Analysis of the amino acid
significantly influenced by the results sequence of he Kat G product has shown that it is
characterized bacterial null mutants. similar to peroxidase, but bears no resemblance to
studicd are the null mu
Among the most intensively and Sod B any of the known catalase sequences'. The third gene.
tants for Mn-SOD
and Fe-SOD (Sod A kut F, is required for expression of kat E, the kat F
(gor genes) and the two
genes), glutathione reductase (kat G and kat E genes). product appears to be a positive regulator of kat E (ref
different forms of catalase genes lacked 125).
carrying mutations in Sod A or Sod B
Cells CURRENT SCIENCE, VOL. 82, NO. 10, 25 MAY 2002

1234
 REVIEW ARTICLES

Use of native plant gene chloroplastic form can be distinguished from the cyto
solic form by its labiality in the absence of ascorbate,
The isolation of native plant genes provides an opportu its high specificity for ascorbate and its narrow pH opti
mum. Four discrete bands of AsAPOD activity can be
nity either to over express the native genes in order to
increase enzyme activity or to produce enzyme separatcd from poplar lcaves by activity staining fol
deficient plants using antisense technology. lowing non-denaturing PAGE. Anion-cxchange chroma
tography has becn uscd to purify AsAPOD, and two
isoforms (AsAPOD I and II) have been detected, iso
Superoxide dismutase form I is a plastidic protein, while isoform II cytosolic.
Several cDNAs that encode eiher chloroplastic or cyto
solic plant CuZn-SOD have been isolated. The cDNAs Glutathione peroxidase
for chloroplastic CuZn-SOD rom tomato26 , pea127 and
a28 peroxidase activity is absent in leaf
and for cytosolic CuZn-SOD of maize129 Glutathione
extracts. Jablonski and Anderson showed that H,0,
and Nicotiana plunbaginifolial have bcen iso dependent oxidation
of glutathione could be measured
lated. The cDNAs for Mn-SOD and Fe-SOD, which are
located in the plant mitochondria and the chloroplast, in pea shoot extracts, but this invoBved more than one
respectively have also been isolated and cloned from N. protein. Selenium-independent glutathione peroxídase
plhunbaginifolia! and Arabidopss thaliang132 Over has recently been identified in higher plants, but they
expression of native foms of SOD in transgenic plants are not normally expressed in leaves or roots. A cDNA
has been achieved in several laboratories, with appar library from freshly isolated Nicotiana sylvestris proto
ently conflicting results. plasts was screened using cDNA from mesophyll cells,
Teppermann and Dunsmuirl33 produced ransgenic stressed leaf strips and cell suspension cultures'42 A
clone with homology to
mammalian selenium
tobacco plants expressing the Cu/Zn-SOD from petunia was isolated.
in addition to the native forms of the cnzyme. The dependent glutathione peroxidase (GPOD)
Selenium-dependent GPOD enzymes are largely absent
transgenic tobacco lcaf discs expressed 30 to 50-fold
more Cu/Zn-SOD than controls. However, they were from higher plants, but selenium-independent GPOD of
unknown function induced under stress condi
protected against methyl-viologen-mediated inhibi
are
not
tions
142
There is no clear evidence from biochemical
tion of "CO, assimilation nor chlorophyl bleaching
during photo-inhibitory conditions130 nor were they pro studies that these GPODlike enzymes have activity in
silu and their function is unknown.
tected against 0Zone toxicityl34 These authors sug
gested that elevating SOD alone could not protect
against Oxygen toxicity and that it would be necessary Peroxidase
to increase simultaneously the enzymes involved in
H,0, detoxification. In contrast, high level of overpro
A gene encoding an anionic tobacco peroxidase (POD)
duction of Mn-SOD leading to protection against
has been expressed in both transgenic tobacco*3 and
methyl viologen has also been reported35,136 ransgenic tomato144 under the control of the CaMV 3SS
promoter. Several physiological processes Were dra
Glutathione reductase matically affected by POD over-production, and severe
wilting was found in transgenic plants.
In comparison to SOD, GR has received little attention.
The genctic relationship between pea GR and that from Catalase
other organisms has been studicdl37 In addition, the
native pea GR gene introduced into transgenic tobacco Peroxisomes contain large amounts of catalase, but its
13
plants, and GR-deficient plants, produced by anti properties suggest that the enzyme is inetticient in
sense technology, is also being studied. removing low Concentrations of H,02. Catalase
deficient mutants and cDNA for different catalase genes
have been isolated and characterized", A barley mutant
Ascorbate peroxidase largely deficient in catalase but with an increased glu
Ascorbate peroxidase has been purified and character tathione content, was unable to survive under photores
and pea lcaves9. ASAPOD piratory conditions, but grew wcll in a high CO
ized from spinach3 tea
is a heme-protein more similar in primary structure to atmosphererel4s In contrast, a maize mutant deficient in
two of the catalase isoforms grew well in air and resem
yeast cytochrome-C POD than to the guaiacol peroxi
bled the wild-type in phenotype"80,146 Photorespiratory
dase, such as horseradish peroxidase, AsAPOD exists
in both soluble cytosolic and chloroplastic forms. The CO) loSs was decreased by 10-20% in a high catalase
1235
CURRENT SCIENCE, VOL. 82, NO. 10, 25 MAY 2002
REVIEW ARTICLES
mutant of N. tabacun var. Havana, with a 40% higher
catalase activity than the wild type". This decrease in
photorespiration was considered to result from inhibi
tion of the chemical decarboxylation of keto acids by
peroxisomal H,02. This interpretation implies that
introduction of cloned catalasc genes into Ca plants may
offer an opportunity to decrcase photorespiratory carbon
loss, since it appears that endogenous catalase at nomal
levels is too low to compete effectively with keto acids
for peroxisomal H;0;.
Conclusions and perspectives
Although oxidative stress is potentially a lethal situa
tion, it is also clear that plant systems cxploit the inter
action with oxygen. The production and destruction of
active oxygen species is intimately involved with proc
esses such as the hypersensitive responses and the regu
lation of photosynthetic clectron flow. The activity of
the antioxidative defence system must be equal to the
task of destruction of reactive oxygen species in normal
metabolism and at times when the plant suffers stress.
However, the antioxidative defence systenm of plants is
quite limited in its capacity to respond to sress, the
activities of component enzymes the antioxidant
levels usually only double in response to many stress
situations. This rather moderate response might be
understood if we consider that the system is geared to
self-destruction when it comes under threat.
We must consider that antioxidants are not always
accessible to some of the sites where they are most
needed in times of stress. Examples of this are the lim
iled availability of ascorbate in the apoplastic space
during attack by the pollutant ozone, the very poor dif
fusion of ascorbate across the thylakoid membrane that
provides ascorbate for the
reaction19,103 and the absence of violaxanthin deepoxidase
effective antioxidants at
the PSI reaction centre to prevent the oxidative damage
associated with photo-inhibition.
Studies carried out by many research groups with
prokaryotic and eukaryotic systems have considerably
supplemented previous pure physiological or biochemi
cal approaches. Genetic enginecring also offer advan
tages in terms of the study of the physiological roles
of enzymes where a classical genetic approach, such
selection of
enzyme-deficient mutants, is difficult
or almost impossible to carry out. In plant systems,
the situation is often considerably
complicated by
the presence of a large number of isoenzyme forms,
for example, the large GR and SOD families of isoen
ymes, cncoded by different genes. In the future, how
ever, the use of antisense technology combined with
selection of specific cDNA clones for isoenzymes may
facilitate investigation of such enzyme-deficient
mutants.
1236
provided
The ability to generate transgenic plants has
a powerful tool with which to increase our present
network.
understandirng of the antioxidative defence
This work extends and compliments similar research
work on prokaryotic systems. From the data accumu
latcd thus far, it is clcar that an appropriate physiologi
cal balance of all he components of the antioxidative
defences is necessary in order to obtain increases in
stress tolerance. Current observations suggest that
incrcasing the lcvel of stress tolerance by reinforcing
the plant's defence systern with new genes is an attain
able goal. In future, better appreciation of control of the
expression of the native genes, increasing the activities
of the cnzymes of the antioxidant systens by manipula
tion of the regulatory processes controlling their expres
sion, may provide an additional mcans of improvement.
1. Fridovich, I.., Annu. Rev. Biochem., 1995, 65.97-112.
2. Alscher, R. G., Donahue, J. L. and Cramer, C. L., Physiol.
Plant., 1997, 100, 224-233.
3. Asada, K., in Causes of Photooxidative Stress and Ameliora
tion of Defence Systems in Plants (eds Foyer, C. H. and
Mullineaux, P. M.), CRC Press, Boca Raton, FL, 1994, pp. 77
104.
4. Bennoun, P., Biochim. Biophys. Acta, 1994, 1186, 59-66.
S. Asada, K. and Takahashi, M., in
Photoinhibition: Topics in
Photosynthesis (eds Kyle, D. J., Osmond, C. B. and Arnten,
C. J.), Elsevier, Amsterdam, 1987, vol.9, pp. 227-287.
6. Gressel, J. and Salun, E., in Causes of Pho tooxida tive Stress
and Amelioration of Defence Systems in Plants (eds Foyer,
C. H. and Mullineaux, P. M.), CRC Press, Boca Raton, FL,
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7. Foyer, C. H., Descourvieres, P. and Kunert, K. J., Plant Cell
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8. Castillo, F. J. and Greppin, H., Environ. Exp. Bot., l988, 26,
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10. Zelitch, I., ibid, 1990, 92, 352-3S7.
11. Zelitch, I, in Perspectives in Biochemical and Genetie Regula
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1990, pp. 239-252.
12. Egneus, H., Heber, U., Matthiesen, U. and Kirk, M.,Biochim.
Biophys. Acta, 1975, 408, 252-268.
13. Heber, U., Egneus, H., Hanch, U., Jensen, M. and Koster, s.
Planta, 1978, 143, 41-53.
14. Wu, J., Nelnanis, S. and Heber, U., Bot. Acta, 1991,104, 283
291
15. Knox, J. P. and Dodge, A. D., Phytochemistry, 198S, 24, 889
896.
16. Cadenas, E., Annu. Rev. Biochem., 1989, 58, 79-110.
17. Mehlar, A. H., Arch. Biochem. Biophys., 1951, 33, 65-77.
18. Harbinson, J. and Hedley, C. L., Plant Physiol., 1993, 103,
649-660.
19. Foyer, C. H., Furbank, R. T., Harbinson, J. and Horton, P.
Photosyn1h. Res., 1990, 25, 83-100.
20. Hartbinson, J., Genty, B. and Foyer, C. H., Plant Physiol.,
1999, 94, 545-553.
21. Badger, M. R., Annu. Rev. Plant Physiol., 1985, 36, 27-53.
22. Asada, K., Kiso, K.. and Yoshikawa, K.,J. Biol. Chen., 1974.
249, 2175-2181.
23. Nakano, Y. and Asada, K., Plant Physiol., 1981, 22, 867-880.
CURRENT SCIENCE, VOL. 82, NO. 10, 25 MAY 2002
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