Sei sulla pagina 1di 35

Abiotic stress in plants: mechanism and management strategies


DR. M Balakrishnan


Shashi Meena
Scientist- trainee
FOCARS-101 st

ICAR-National Academy of Agricultural Research Management

Rajendranagar, Hyderabad-500030, Telangana, India

Introduction----------------------------------------------------------------------------------- 3-4
Sources of ROS--------------------------------------------------------------------------------4-6
Types of ROS---------------------------------------------------------------------------------6-10
Superoxide radicals (O2.-)
Singlet oxygen (1O2)
Hydrogen peroxide (H2O2)
Hydroxyl radical (OH.)
Damages due to ROS----------------------------------------------------------------------10-16
Lipid peroxidation
Protein oxidation
DNA damage
Conditions enhancing overproduction of ROS----------------------------------------16-19
Metal toxicity
High temperature
ROS scavenging mechanism in plants------------------------------------------------19-20
Non-enzymatic antioxidants----------------------------------------------------------20-28
Ascorbic acid (Vitamin-C)
Glutathione (GSH)
Tocopherol (Vitamins-E)
Phenolic compounds
Enzymatic antioxidants----------------------------------------------------------------28-33
Superoxide dismutase (SOD)
Catalase (CAT)
Guaiacol peroxidase (GPOX)
Glutathione reductase (GR)
Monodehydroascorbate reductase (MDHAR)
Dehydroascorbate reductase (DHAR)
Ascorbate peroxidase (APX)
Glutathione-S-transferase (GST)
Glutathione peroxidase (GPX)

Abiotic stress in plants: mechanism and management strategies


Introduction:Plants grow and reproduce in a complex environmental conditions composed of a multitude

of abiotic (non- living) and biotic (living) factors. These abiotic stress factors are naturally
occurring, often intangible, such as light intensity and quantity, temperature (high and low),
drought, salinity, chilling. These abiotic factors vary both in time and with geographical
location. Abiotic stress is defined as the negative impact of non-living factors on the living
organisms in a specific environment. The non-living variables must influence the
environment beyond its normal range of variation to adversely affect the biochemical and
physiological functions of the plants in a significant way. The biotic factors include
pathogen/pest attack on plants.Abiotic stress is essentially unavoidable. Abiotic stress also
affects animals, but plants cannot physically move away from environmental factors, so it is
particularly constraining. Abiotic stress is the most harmful factor concerning the growth and
productivity of crops worldwide. The plants which grow under these unfavourable
environmental conditions have negative impacts and various mechanisms to tackle these
conditions. To survive under these conditions, plants have to develop a complex signalling
network including different endogenous growth regulators that acts as a sensor. One of the
common mechanisms is the accelerated generation of reactive oxygen species (ROS) under
both biotic and abiotic stresses (Bhattarcharjee, 2011). ROS is produced in plants under
various normal metabolic processes (Fridovich, 1995). But the balance between production
and antioxidative system is disturbed under both abiotic and biotic stress conditions. ROS
production is the key molecule during stress in plant tissues.
The ROS free radicals are also produced under various stress conditions in different cellular
compartments, including chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum,
plasma membrane (Mittler, 2002; Asada, 2006; Navrot et al., 2007). When the level of ROS
exceeds the antioxidants, then a cell is said to be in a state of oxidative stresses. Oxidative

burst is the result of ROS production either extracellular or intracellular, which has negative
impact on physiological and biochemical functions of the plants (Gill et al., 2010). Plants
must be able to sense the environmental cues through sensors before being able to respond
appropriately to the abiotic stress. A signal transduction pathway invoked after initial
recognition of stress which ultimately result in the production of secondary messenger,
involved in the activation of stress induced genes. Under normal conditions ROS act as
signalling molecule but under stress condition it results in oxidative stress or burst in cell.








Fig: Main sources of ROS in plant cell

Ground state O2 is relatively unreactive and this is the precursor molecules which give rise to
ROS during both normal metabolic activity and various environmental perturbations.
Reactive oxygen species (ROS) comprises both free and non-free radical oxygen species
including superoxide (O2.-), perhydroxy radical (HO2), hydrogen peroxide (H2O2), hydroxyl
radical (OH.), alkoxy radical (RO), singlet oxygen (1O2) (Bhattarcharjee, 2011).Chloroplast is
the major source of singlet oxygen (1O2) associated with electron transport chain. This active
oxygen in plant tissues may also arise as a by-product of lipid peroxidation, which is

catalysed by enzyme lipoxygenase (LOX) (Asada, 2006; Foyer et al., 2009). Due to stress
conditions, there is limited CO2 fixation by Calvin cycle leads to a decrease in oxidized
NADP+ which serves as electron acceptor in photosynthesis. Stress conditions lead to
overloading of the ETC, result in leakage of electron from PSI (Photosystem I) to O 2,
reducing it to O2.-. This process is known as Mehler reaction (Karunppanapandian et al.,
2011). One of the major problems that plants encounter during stress is the uncoupling of the
light reaction of photosynthesis from the fixation of CO2 and production of highly reactive
chlorophyll molecules which ultimately result in the production of highly reactive singlet
oxygen species.
ROS production in peroxisome is the result of their essentially oxidative pathways and is the
major site of intracellular ROS production. Peroxisomes also produce superoxide radicals
through normal metabolism. Glycolate oxidase is the enzyme of photorespiratory pathway
(C2) in plants which involved in the oxidation of glycolate to glyoxylate and also result in the
production of H2O2 (Mittler et al., 2004). There are two sites of O2.-generation in peroxisomes
(del Rio et al., 2002). One site is located in the organelle matrix, in which enzyme xanthine
oxidase (XOD) is present and another site is located in the peroxisomal membranes which
have NAD(P)H-dependant ETC. Xanthine oxidase catalyzes the oxidation of xanthine and
hypoxanthine to uric acid and a well-known producer of superoxide radicals (Lopex-huertus
et al., 1999; del Rio et al., 2002). The apoplast is alsoan important site for H2O2 production
in plants under adverse environmental conditions such as drought and salinity (Zhu et al.,
2001). The two enzymes are involved in apoplastic ROS production such as oxalate oxidase
and amine oxidase (Mittler, 2002), catalyse the oxalic acid and causes oxidative deamination
of ployamines (i.e. putrescine, spermine, and spermidine) respectively (Cona et al., 2006).
The various electron transporting oxidoreductases such as NADPH oxidase and quinine

reductase are present at plasma membranes and lead to production of free radicals at plasma
membrane under stress conditions (Heyno et al., 2011).
ROS production in mitochondria takes place under normal respiratory conditions but can be
enhanced in response to various environmental stress conditions. The primary source of
mitochondrial ROS generation is the mitochondrial ETC, which harbours electrons with
sufficient energy to directly reduce O2 (Rhoades et al., 2006). The secondary source of ROS
production in mitochondria is mitochondrial matrix which contains several enzymes. Cell
walls are the major sites for active metabolism and ROS production in plants. NADH oxidase
localised to the plasmalemma and involved in defence mechanism against pathogens and
pests. NADH oxidase give rise to H2O2 catalyzing NADH, generated by cell wall associated
malate dehydrogenase enzymes (Higuchi, 2006). Diamine oxidase, Lipoxygenase and cell
wall localised peroxidases are also involved in the production of activated O 2 during stress
conditions. The endoplasmic reticulum is also involved in superoxide radical generation
through NAD(P)H- dependent electron transport involving cytochrome P450 (Mittler, 2002).
Types of ROS:Chloroplast, mitochondria or peroxisomes are the organelles with highly oxidizing metabolic
activity or with intense rate of electron flow. These two activities are major sources of ROS in
plant cells (Gill et al., 2010). Plants have ability to use O2 for the energy production and this
energy is utilized for their own developmental processes. It has been estimated that 1-2% of
O2 consumed by plants is contributed to ROS production in various subcellular loci (Blokhina
et al., 2003). ROS is derived from ground state oxygen either by energy transfer or by
electron leakage reactions. The former reaction leads to the formation of singlet oxygen ( 1O2),
whereas the latter reaction results in the sequential reduction to superoxide (O 2.-), hydrogen
peroxide and hydroxyl radical (Klotz, 2002). In plants active oxygen species are continuously

produced as by-products of various metabolic pathways localized in different cellular

compartment (Maxwell et al., 1999). There is very high risk of oxidative damage to
photosynthesizing organisms, because of the abundance of photosensitizers and
polyunsaturated fatty acids (PUFA) in the chloroplast envelope. The other reason of oxidative
damage is their bioenergetic lifestyle (Gill et al., 2010).
Aerobic respiration in plants is also other source of ROS generation (Temple et al., 2005).
O2.- is generated by single electron reduction of O2. At low pH, O2.- may give rise to peroxide
ion by simply added electron to it and then with protonation resulting in the generation of
H2O2. O2.- can be protonated to form OH.. H2O2 further react with transition metals such as
copper and iron result in the formation of OH. . This reaction is called the Fenton reaction or
the Haber-Weiss mechanism. OH. is the most reactive oxygen species in the biological world.
Singlet oxygen is another form of ROS; it can be formed by photoexcitation of chlorophyll
and its reaction with O2. This is the case there is no addition of an extra electron to O 2. O2.can also react with another signalling free radical species, NO. give up peroxynitrate (OONO-)
(Gill et al.,2010).
Dioxygen e(3O2)
Singlet oxygen

Superoxide radical
ion (O2.-)


peroxide eion (O22-)



ion (O-)


l radical



Fig: Generation of different ROS by energy transfer or sequential univalent reduction

of ground state triplet oxygen (Apel et al., 2004)
Superoxide radicals (O2.-):- Superoxide radical is produced in plant cell at the onset
of oxidative burst of cell. O2.- is produced by a single electron reduction of O 2. O2.- is
a moderately reactive and have a short lived ROS with a half-life of approximately
1s ( Gill et al., 2010). It is easily dismutated to produce H 2O2. This process is

carried out either spontaneously or by superoxide dismutase (SOD). Furthermore, O 2.can also donate electron to Fe3+ to yield a reduced form of iron (Fe 2+). This reduced
form of iron (Fe2+) oxidized by H2O2 and rapidly generate OH. . This reaction is the
Fentons reaction or the Haber-Weiss reaction.
The generations of superoxide radicals also trigger the production of more reactive
ROS like OH. and 1O2. These reactive oxygen species may cause membranes lipid
peroxidation and cellular weakning. The major site of superoxide radical formation is
the thylakoid membrane bound primary electron acceptor of PSI during
photosynthesis. It is also produced during aerobic respiration (Arora et al.,2002).
1. Singlet oxygen:-1O2 is an unusual form of ROS in plants. It can be produced by
insufficient energy transfer during photosynthesis to chlorophyll, result in the
formation of triplet state chlorophyll (3Chl*). This triplet state can react with dioxygen
(3O2) to give up the very reactive 1O2. For the formation of 1O2, electron transfer to O2
does not require. The life span of singlet oxygen within the cell is approximately 3s
(Hatz et al., 2007; Hackbarth et al., 2010). It has been found that a fraction of 1O2 has
been able to diffuse over considerable distances of several 100 nanometers (nm). It
has been also found that 1O2 can last for 4s in water and 100s in a non-polar
environment (Foyer et al., 1994). 1O2 reacts with most of the biological molecules and
it directly oxidizes unsaturated fatty acid, protein and DNA (Foyer et al., 1994). It is
documented to be the most reactive species responsible for light-induced loss of
Photosystem II (PSII) activity which may trigger cell death (Liszkay et al., 2008). It is
well established that if 1O2 is produced through normal metabolic pathways, it is
efficiently quenched by -carotene, tocopherol or plastoquinone. If it is not quenched
by these molecules, then it can activate the upregulation of genes, involved in the
molecular defense responses against photo-oxidative stress (Liszkay et al., 2008;
Lizskay, 2005).

2. Hydrogen peroxide (H2O2):- H2O2 is generated in the cells under non-stressed as

well as stressed conditions. There are various major sources of H2O2 generation in
plant cells including electron transport chain (ETC) of chloroplast, mitochondria, ER
and plasma membrane, -oxidation of fatty acid in glyoxysome and photorespiration
in peroxisome (Sharma et al., 2012). H2O2 is produced by univalent reduction of O2.-.
H2O2 is moderately reactive and has relatively long half-life (1ms). It can diffuse some
distances from site of production through aquaporins in the membrane (Halliwell,
2006; Moller et al., 2007; S. Bhattacharjee et al.,2007). Under stress conditions, the
amount of H2O2 is enhanced and it may inactivate enzymes by oxidizing their thiol
groups. OH. is the most reactive oxidant in the family of ROS, H 2O2 can also involved
in the generation of hydroxyl radical (OH. ) through Haber-Weiss/ Fenton reactions.
H2O2 is also produced by the dismutation of superoxide radicals either spontaneously
of by superoxide dismutase (SOD).
H2O2 plays a dual role in plants:- At low concentration, it acts as a signal molecule
which involved in the regulatory mechanism of specific biological process and
triggering tolerance to various biotic and abiotic stresses (Torres et al., 2002). At high
concentration, it can oxidized the cysteine (-SH) or methionine residues (-SCH 3), thiol
groups of enzyme such as enzymes of calvin cycles Cu/Zn-SOD and Fe-SOD
(Halliwell and Gutteridge, 1989). At high concentration it leads to PCD (Quan et al.,
3. Hydroxyl radical (OH.):- OH. radicals are the most reactive among all ROS. This
ROS can be formed from O2.- and H2O2(Bhattacharjee, 2010). It is highly reactive
ROS and it can interacts with all biological molecules and causes oxidation of DNA,
proteins, lipids, and almost any constituent of cells. Plant cells does not have efficient
mechanism to scavenge this highly reactive oxygen species, it production in excess
results in programmed cell death (PCD) (Karuppanapandian et al., 2011; Foyer et al.,

1997). OH. is also responsible for the oxidation of organic substrates, may proceed by
two possible reactions either by addition of OH. to an organic molecule or by
abstraction of a hydrogen atom from it. These two reactions which involve oxidation
of organic substrates, also leads to the production of destructive free radicals such as
alkoxy, peroxy, semiquinones, reduced H2O2 etc., besides toxic OH. (Arora et al.,
Damages due ROS:In order to avoid oxidative damages or stress, the production and removal of ROS must be
strictly regulated. Under various stressful conditions such as drought, salinity, high light,
metal toxicity, temperature and uv-radiation, the balance between production and scavenging
of ROS is perturbed. Oxidative stress is the condition in which the level of ROS exceeds
the level of antioxidants in the cell. High level of ROS can cause damage to various
biomolecules such as lipids, DNA and proteins. ROS not only affect these molecules, it can
also alter intrinsic membrane properties like ion transport, fluidity, inactivation of enzyme,
cross-linking of protein, inhibition of protein synthesis, damage to DNA, etc. ultimately
resulting in autophagy (cell death).
Fig: The graph showing relationship between

antioxidants and stress.


damage 10



Alter membrane


Damage to

Fig:- Abiotic stress induced ROS production and cell death.

Lipid peroxidation:- Under stressful conditions, the production of ROS reaches to

high level, in turn it affect normal cellular functioning by enhancing lipid
peroxidation. Lipid peroxidation is the most damaging process known to occur in
every living organism. Oxidative stress condition result in the production of lipid
derived radicals that themselves can react and damage DNA and proteins. It has been
found that the plants growing under environmental stresses result in increased
degradation of lipids and also with increased production of ROS. There are several
reactive molecules such as ketones, MDA (Malondialdehyde), lipid epoxides etc,
formed from polyunsaturated precursor during LPO (Halliwell et al., 1989).
LPO process involves 3 distinct stages:-

Initiation:- This steps also involves transition metal complex, especially those of Fe
and Cu. O2.- when react with H2O2 give rise to OH., highly reactive. This free radical
initiates the lipid peroxidation in a membrane by reacting with a polyunsaturated fatty
acid (PUFA). This reactions result in the formation of lipid alkyl radical (R.). This
further converted into ROO.- (lipid peroxy radical) in aerobic condition. A single
initiation event has the potential to generate multiple peroxide molecules by a chain
reaction (Gill et al., 2010).

Initiation step:RH + OH.

R. + H2O


(Lipid Alkyl radical)

Propagation:- The peroxy radical formed is highly reactive and able to propagate the
chain reaction.
Propagation steps:R. + O2

(Lipid peroxy radical)


RO.Epoxides, hydroperoxides, glycol, aldehydes


Termination:- The LPO reaction stopped when two free radicals combine together
and form fatty acid dimer.
Termination steps:R. + R.

(Fatty acid dimer)

R. + ROO.

(Peroxide bridged dimer)


(Peroxide bridged dimer)

Lipid peroxidation events decreases membrane fluidity, make it leaky, damage membrane
proteins, inactivation receptors, enzymes, ion channels (Moller et al., 2007). PUFA

peroxidation result in the production of MDA and HNE (2-hydoxy-2-nonenal), from linolenic
and linoleic acids respectively.

Fig:- PUFA peroxidation

PUFA peroxidation also results in the production of hydroxyl and keto fatty acids. The
breakdown products of peroxidation react with DNA and proteins (Moller et al., 2007). It has
also been found that plants exposed to various biotic and abiotic stresses exhibit an increase
in LPO due to generation of ROS (Singh et al., 2008). Kukreja et al., 2005 observed that
marked increase in LPO in Cicer arientinum roots under salinity stress.

Protein oxidation:-ROS production under biotic and abiotic stresses, does not only
initiates LPO but also result in covalent modification of proteins. Protein
conformation and stability is dramatically affected by sudden changes in the
environment, giving rise to protein unfolding, misfolding and aggregation. Folded
states represent the most stable forms under native conditions, but partially folded
states that allow for efficient interaction with binding partners are of fundamental
importance in biological activity. Protein function is dependent on its unique threedimensional structure that is adopted by the initial folding of the polypeptide chains
after translation. Folded proteins are generally much less prone to aggregation and

degradation but partially unfolded or intrinsically disordered regions of proteins can

confer functional advantages, as they allow efficient interaction with binding partners
and provide a mechanism for the regulation of cellular processes, hence they are
highly susceptible to reactive oxygen species. Some of the protein modifications are
irreversible, whereas a few involving sulfur-containing amino acids are reversible
(Ghezzi et al., 2003). Protein oxidation may be direct and indirect. Direct
modification of protein involves covalent modification of proteins activity through
several processes like nitrosylation, carbonylation, glutathionylation and disulphide
bond formation. Indirect modification of protein involves conjugation with
breakdown products of fatty acid peroxidation (Yamauchi et al., 2008). Carbonylation
of protein is widely used marker of protein oxidation (Moller et al., 2007). As a
consequences of excessive ROS production, the oxidation of a number of protein
amino acids particularly Arg, His, Lys, Pro, Thr, and Typ gives rise to free carbonyl
groups which may inhibit or alter their activities, fragmentation of the peptide chain,
altered electric charge and increased susceptibility of proteins towards proteolytic
attack (Moller et al., 2007). ROS has ability to attack on certain amino acids in a
peptide. Thiol groups and sulphur containing amino acids are very susceptible sites
for attack by ROS. Cysteine and Methionine are quite reactive especially with 1O2 and
OH.. Activated oxygen can abstract hydrogen atom from cysteine residues to form a
thiol radical that will cross-link to a second thiyl radical to form disulphide bridges. In
the presence of ROS, tyrosine is readily cross-linked to form bityrosine product
(Davies, 1987) and methionine to form methionine sulphoxide derivative (Brot and
Weissbach, 1982). Sarvajeet Singh Gill and Narendra Tuteja, 2010 noted that various
abiotic and biotic stresses lead to the carbonylation of proteins in tissues and proteins


can be damaged in oxidative conditions by reacting with LPO products, such as HNE
and MDA.

DNA damage:- DNA is the genetic material of the cell and any damage to the DNA
can result in malfunctions or complete inactivation of encoded proteins. DNA can be
damaged by ROS production under biotic and abiotic stresses. High level of ROS not
only damage to cell structures, lipids and proteins but also damage nucleic acid (Valko
et al., 2006). ROS can cause oxidative damages to nuclear, mitochondrial, and
chloroplastic DNA. Nuclear DNA is more resistant to oxidative damage than
mitochondrial and chloroplastic DNA due to the presence of protective proteins and
histones (Richter, 1992). High level of ROS or oxidative stress attack on DNA results
in deoxyribose oxidation, removal of nucleotides, strand breakage, modification of
bases and DNA protein crosslinks, also enhance the chances of mutations (causes
G:C alterations).ROS attacked on both sugar and base moieties of DNA. ROS attack
on DNA bases generally involves OH .addition to double bond, while sugar damage
mainly involves hydrogen abstraction from the deoxyribose. It has been reported that
OH. is most reactive radical and causes damage to purine and pyrimidine bases and
also deoxyribose backbone (Gill et al., 2010). Upon reaction with DNA, it results in
the production of C-8 hydroxylation of quinine to form 8-oxo-7,8 dehydro-2deoxyguanosine etc (Tsuboi et al., 1998). 8-hydroxyguanine is the most commonly
observed product during oxidative stress. Singlet oxygen is dangerous to guanine
only, whereas H2O2 and O2.- do not react with bases at all. ROS can indirectly attack
DNA bases by producing reactive molecules from lipid peroxidation. When the plants
are exposed to various environmental stresses such as salinity (Liuet al., 2000) and
metal toxicity (Meriga et al., 2004) enhances DNA degradation. ROS attack to DNA
sugars result in single-strands breaks. It has been reported that DNA protein cross

links formed when OH. attacks on either on DNA or protein associated with it. DNA
damage affects various physiological functions of plants (Gill et al., 2010).
Conditions enhancing overproduction of ROS:Under normal growth conditions, the production of ROS in plants is low. However, in
response to various stress conditions, the generation of toxic O2 species is drastically
enhanced in plants disturbing the balance between production and scavenging of O2.-,
OH. and H2O2 in the intracellular environment (Sharma et al., 2010). The effects of
various stress factors such as drought, salinity, chilling, metal toxicity, ROS
production are described below:1. Drought:- ROS production in plant is enhanced under drought stress by several
ways. Several activities under drought stress results in overproduction of ROS
through the chloroplast Mehler reaction (Asada, 1999). During drought stress, there
is inhibition of CO2 assimilation which in turn, leads to reduced NADP+ generation
through the C3 cycle. This result in lack of NADP+ electron acceptor, over reduction
of the photosynthetic ETC occurs which leads to a higher leakage of electrons to O 2
by the Mehler reaction (Sharma et al., 2012). Under drought stress, there is an
imbalance between light capture and its utilization, this result in decreased in
photosynthetic activity in plant tissues (Foyer et al., 2000). The other reason of
ROS generation is dissipation of excess light energy in the PSII core and antenna,
which are potentiallly dangerous under drought stress. Under drought stress, the
photorespiratory pathway is also enhanced and there is more RUBP oxygenation
due to limitation in CO 2 assimilation (Noctor et al., 2002). Sangmein lee and
Chung-MO Park found that the NTL4 gene is induced in the response of H 2O2, and
also related with the accumulation of ROS under drought stress. Under drought
stress conditions, 70% of total H2O2 production is contributed by photorespiration

(Noctor et al., 2002). Enhanced production of ROS leads to oxidative stress in

growing plants.
2. Salinity:- High salt concentrations results in an excessive generation of ROS by
impairment of the cellular electron transport within different subcellular
compartments such as chloroplasts and mitochondria, as well as induction of
metabolic pathways such as photorespiration. Salt stress can lead to stomatal
closure, low chloroplastic CO2/O2 ratio also favors photorespiratory pathway
leading to increased production of ROS such as H2O2 (Hernandez et al., 2000).
During salt stress, less CO2 available in the leaves leading to enhanced generation
of ROS and induced oxidative stress due to exposure of chloroplasts to excessive
excitation energy and overreduction of photosynthetic electron transport system
(Hernandez et al.,

2000). Salinity induce disruption in normal subcellular

metabolism through lipid peroxidation, denaturing proteins and nucleic acids in

several plant species by the production of ROS (Hernandez et al., 2000). Elevated
CO2 mitigate the oxidative stress caused by salinity. Salt-sensitive cultivar seedlings
showed a substantial increase in the production of O2.- elevated levels of H2O2,
MDA, declined levels of thiol, ascobate and glutathione and lower activity of
antioxidant enzymes compared to salt- tolerant seedlings.
3. Chilling:- This factor leads to the overproduction of ROS by disrupting the balance
between light capture and light assimilation by inhibiting Calvin-Benson cycle
activity (Bouraoui et al., 2011). This factor also enhances photosynthetic electron
flux to O2 and causing overreduction of respiratory ETC (Hu et al., 2008). Chilling
stress also causes significant reductions in rbcL and rbcS transcripts. This will result
in significant reduction in RUBISCO content and initial activity of RUBISCO,
leading to higher electron flux to O2 (Zhou et al.,2006). During this stress, there is
significant accumulation of ROS, including H2O2 and superoxide radical thus leads


to reduction in growth and productivity of crop plants (Fryer et al., 1998; Prasad,
1997; Zhang et al., 2008).
4. Metal toxicity:- Metals are essential for functioning of physiological and
biochemical processes and consequently for normal growth and development of
plants. The increasing level of metals into the environment has negative impact on
plant growth and metabolism, ultimately leading to reduce crop yields (Salt et al.,
1995; Mishra and Dubey, 2005). Net photosynthesis decreases due to damage to
photosynthetic metabolism, including photosynthetic electron transport under metal
stress conditions. Alter photosynthetic metabolism lead to overproduction of ROS.
Metal stress affects various mechanisms: such as interference with functional sites
in proteins, displacement of essential elements, thereby disturbing enzymatic
functions, enhanced ROS production. Heavy metal stress causes various problems
in plants:- Induction of peroxidation which can directly cause biomembrane
deterioration, decomposition of polysaturated fatty acids (PUFAs) result in the
production of oxidative stress (Karunppanapandian and Manoharan, 2008;
Karunppanapandian et al., 2009, 2011).
5. High temperature:- Temperature stress is one of the major type of abiotic stress
and has devastating effects on plant growth and metabolism. High temperature is
the result of global climate change and a critical factor for plant growth and
productivity; high temperature is now considered as major stresses for restricting
crop production. The growth and development of plants involves a large number of
biochemical reactions, all of which are sensitive to some degree to temperature.
Consequently, plant responses to high temperature vary with the extent of the
temperature increase, its duration, and the plant type. When plants exposed to high
temperature stress leads to excess accumulation of toxic compounds, especially
reactive oxygen species (ROS). In response to high temperature, the reaction


catalyzed by RUBISCO can lead to the production of H 2O2 as a consequence of

increase in its oxygenase reaction. Under HT condition, the stomatal
conductancealso decreased significantly. Prasad et al. reported that high night







photosynthetic rate by 8% and 22%, respectively, compared to optimum night

temperature. Deactivation of RUBISCO is one of the causes associated with the
decline in photosynthesis under HT. Many authors reported that the heat-induced
deactivation of RUBISCO is the primary constraint for photosynthesis at
moderately HT and showed that Chl fluorescence signals from PSII are not affected
by temperatures that cause significant deactivation of RUBISCO.
ROS Scavenging mechanism in plants:Plants are also produced ROS by normal cellular metabolic pathways. But its
production is enhanced when plants faces unfavourable environmental conditions
such as metal toxicity, drought, water logging, air pollutants, nutrient deficiency,
salt-stress, etc. The free radicals produced under these stressed conditions are
controlled by various enzymatic and non-enzymatic antioxidative systems.
Different cellular compartments have different ROS scavenging pathways which
are coordinated (Pang and Wang, 2008). ROS can cause oxidative damage to
DNAs, proteins and lipids. To prevent oxidative damages, plant possesses very
efficient scavenging systems against ROS. These defense systems are located both
intra- and extracellular compartment.
Non-enzymatic antioxidants include Ascorbic acid (AA), Glutathione (GSH),
Tocopherols (TOCs), Carotenoids, Phenolic compounds, Proline and Enzymatic
antioxidative system include Catalase (CAT), Ascorbate peroxidase (APX),









(DHAR), Glutathione reductase (GR),



Glutathione-S-transferases (GST),

Glutathione peroxidase (GPX) (karunppanapandian et al., 2011).

Non-enzymatic antioxidants:1. Ascorbic acid (Vitamin-C):- Ascorbic acid is the most abundant, powerful,
low molecular weight and water soluble antioxidant and present in
chloroplasts, cytosol, vacuole and apoplastic space of cells in high
concentration (Foyer et al., 1991). It has a key role in defense against
oxidative stress caused by increased level of ROS. More than 90% of ascorbic
acid is localized in cytoplasm but a small portion of it is exported to the
apoplast. In apoplast it is present in millimolar concentration and here it
represents the first line of defense against oxidative stresses (Hernandez et al.,
Under normal physiological conditions, ascorbic acid mostly present in the
reduced form in chloroplast whereas it act as a cofactor of enzyme
violoxanthin de-epoxidase, thereby dissipating excess excitation energy
(Smirnoff, 2000). Ascorbic acid as considered powerful antioxidant because it
has ability to donate electrons in various enzymatic and non-enzymatic
reactions, thus ascorbic acid is the main ROS-detoxifying compound in the
aqueous phase. Ascorbic acid has central role in several physiological
processes in plants, including metabolism, differentiation and growth.
Therefore, these are mostly found in abundant amount in photosynthetic
tissues. Critical macromolecules protected from oxidative damage by
enhancing the level of Ascorbic acid. Ascorbic acid also provide protection to
the membrane by regeneration of TOC from tocopheroxyl radical (TOC)


(Smirnoff, 2000) by directly reacting with O2.-, H2O2 and by preserving the
enzyme activities which contain prosthetic transition metal ions (Noctor and
Foyer, 1998). H2O2 is removed from the cells through Ascorbate-Glutathione
cycle (Pinto et al.,2003). The major pool of ascorbate is contributed by
Smirnoff- Wheelar pathway(Smirnoff, 2000).
Oxidation of ascorbic acid occurs in two sequential steps:- By producing
monodehydroascorbate (MDHA) either it may re-reduced to ascorbate or it
may disproportionates into ascorbic acid and dehydroascorbate (DHA). In the
Asada-Halliwell pathway (H2O2 scavenging pathway), two molecules of
ascorbic acid are utilized by ascorbate peroxidase to reduce H 2O2 to water and
MDHA. MDHA if not rapidly converted into ascorbate, it can spontaneously
dismutate into DHA and ascorbate and it is reduced to ascorbate by
Monodehydroascorbate reductase which is NAD(P)H-dependent enzyme.
DHA is highly unstable at pH values greater than 6.0 and is decomposed to
tartarate and oxalate (Noctor and Foyer, 1998). DHA is also contributed to
ascorbic acid pool by the enzyme dehydroascorbate reductase which utilized
reducing equivalents from Glutathione (Asada, 1996).
The biosynthesis of ascorbate within the chloroplast provides a putative
mechanism for the regulation of electron transport. Ascorbate is not only a
potent antioxidant, but it is also involved in pH-meditated modulation of PS II
activity and its down regulation is associated with Zeaxanthin formation
(Neubauer and Yamamoto, 1992). This is a potent mechanism for preventing
photo-oxidation. It has been estimated that the level of Ascorbic acid is alter in
various biotic and abiotic stresses ( Mishra et al., 2011). The level of ascorbic
acid is enhanced by overexpressing the enzymes involved in ascorbic acid
biosynthesis confers abiotic stress tolerance (Chaves et al., 2002). But,it has


also been observed that in the roots and nodules of Glycine max under Cd
stress result in decrease in the ascorbate. Cd stress also decrease the Ascorbate
in Cucumin sativuschloroplast and in the leaves of A. thalianaand P.sativum(Li
et al., 2010).
2. Glutathione (GSH):- GSH is an abundant tripeptide (-glutamyl cystein
glycine) of low molecular weight non-protein thiol in plant tissues that plays
an important role in intracellular defense against oxidative damage induced by
ROS. In plant tissues it occurs abundantly in reduced form. It is localized in all
cell compartments like cytosol, vacuole, chloroplast, endoplasmic reticulum,
peroxisome, mitochondria as well as in apoplast (Mittler et al, 1992), here it
perform several physiological functions like detoxification of xenobiotics,
sulfate transport regulation, signal transduction and regulate the expression of
stress responsive genes. It also plays an important role in diverse biological
events including cell division, cell differentiation, cell death and senescence,
pathogen resistence, synthesis of proteins and nucleic acids, synthesis of
phytochelatins for metal chelation and enzymatic regulation (Foyer et al.,

ate reductase

Dehydroascorbate +Glutathione (GSH)

Glutathione (GSSG) + NADPH

Ascorbic acid + Glutathione (GSSG)


Glutathione (GSH) + NADP


It protects the cell from ROS-induced oxidative damage by acting as a free

radical scavenger of O2.-, OH. and H2O2. It can also protect macromolecules
(i.e. DNA, lipid and proteins) either by acting as proton donor in the presence
of organic free radicals or ROS yielding GSSG (oxidized) or by
glutathiolation, It is the process of forming adducts directly with reactive
electrophiles (Asada, 1994). Additionally, it can play a key role in the
generation of other water soluble antioxidant like ascorbic acid through the
Ascorbic acid-Glutathione pathway (Halliwellet al., 1976). It also takes part in
the production of ascorbate from DHA by the DHAR enzyme. Along with
ascorbate, oxidized glutathione (GSSG) is also produce which is further
converted into reduced glutathione (GSH) by the NAD(P)H-dependent
glutathione reductase (GR) enzyme.
The pool of reduced glutathione (GSH) is maintained either by de novo
synthesis or through recycling by GR. GSH is a potent antioxidant and used as
a stress marker to evaluate the stress conditions. GSH synthesis takes place in
the chloroplast and cytosol of plant cells by two compartment specific
isoforms i.e. glutamate-cysteine ligase and glutathione synthetase. The balance
between the GSH and GSSG is a central component in maintaining cellular
redox state (Foyer et al., 2005). GSH also plays a major role during heavy
metal stress because glutathione is a precursor of phytochelatins and also
protect the photosynthetic apparatus from oxidative damage induced by ROS.
The plants with low level of GSH were highly sensitive to even low levels of
Ca2+ were observed by Xiang et al (Xiang et al.,2001).
3. Tocopherol (Vitamin-E):- Tocopherols is the important constituents of
biomembranes and also lipophilic antioxidants. It is considered as potential
antioxidants against ROS and lipid free radicals (Hollander et al., 2005). It


performs both antioxidant and non-antioxidant functions. Generally, they are

considered as antioxidants for membrane stability protection by physically
quenching and chemically reacting with O 2 in chloroplast, thus protecting the
structure and function of PSII (Igamberdiev et al., 2004). There are four
isomers of tocopherols (, , , ) in plants which have different antioxidant
activity due to the methylation pattern and number of methyl groups attached
to the phenolic ring of the polar head structure. Out of these four isomers, only
one i.e. -tocopherol has the highest antioxidative activity due to the presence
of three methyl substituents in its molecular structure. These antioxidants are
synthesized by photosynthetic organisms and are located only in green parts of
plants. Tocopherol is a chain-breaking antioxidant in lipid peroxidation
(autooxidation) which makes it an effective free radical trap. It has been found
that one molecule of tocopherol can quench 220 molecules of singlet oxygen
(1O2) in vitro before being degraded (Fukuzawa et al., 1982). -tocopherol can
react with derivative of PUFA oxidation and gives rise to TOH .by donating
hydrogen atom to lipid radicals (Igamberdiev et al., 2004). Conversion of
TOH. back to -tocopherol (reduced form) by reacting with GSH and ascorbic
acid (Fryer, 1992) or coenzyme Q (Kagan et al., 2000).
The tocopherol biosynthesis occurs in chloroplast and homogenitisic acid
(HGA) and phytyl diphosphate (PDP) as precursor. At least 5 enzymes are
involved in the biosynthesis of tocopherols (Zhou et al., 2010). -tocopherol is
synthesized from -tocopherol in chloroplast with the help of the enzyme tocopherolmethyltransferase (-TMT; VTE4). The plants can be protected
from the harmful effects of ROS or organic free radicals by upregulating the
expression level of enzymes involved in Halliwell-Asada cycle and in
tocopherol biosynthetic pathway.

4. Carotenoid:- Carotenoid formation is one of the mechanism used by plants to

get rid of excess ROS production in photosynthetic organisms. Carotenoids
are lipophilic organic antioxidants located in plastids of photosynthetic
organisms as well as also found in microorganisms. They have ability to
detoxify various forms of ROS (Young, 1991) and provide oxidative stress
tolerance. Carotenoids carry out four major functions in plants to protect from
ROS induced oxidative damage. First, carotenoids absorb light at wavelength
between 400 and 500nm of the visible spectrum and transfer the captured
energy to the chlorophyll (Sieferman-Harms, 1987). Second, they act an
antioxidant, by scavenging 1O2, triplet sensitizer (3Chl) and excited chlorophyll
molecule and protect the photosynthetic apparatus from oxidative stress
(Collins, 2001; Vallabhaneni et al., 2008). Third, they also serve as precursors
to signalling molecules which plays an essential role in plant development and
in biotic/abiotic stress responses. Fourth, they scavenge, prevent or minimize
the synthesis of triplet chlorophyll and other harmful ROS species. In this way,
they are important for protection of the PSI assembly and also maintained the
stability of light harvesting complex proteins as well as thylakoid membrane
(Niyogi et al., 2001).
Zeaxanthin is another form of carotenoid which protects the photosynthetic
apparatus from active chlorophyll molecules by dissipation of thermal energy,
but the precise mechanism for thermal energy dissipation by zeaxanthin is not
resolved (Mortensen et al., 2001). High carotenoid concentration in sugarcane
plants favors better adaptation under saline conditions observed by Gomathi
and Rakkiyapan. Carotenoids are able to dissipate excess energy from excited
molecules due to the presence of numerous conjugated double bonds.


5. Phenolic compounds:- Phenols also protect plants form toxic ROS. These are






hydroxycinnamate, ester and lignin, which possess antioxidant properties.

These secondary metabolites are abundantly present in the plant kingdom and
are commonly found in various parts of the plant kingdom including in leaves,
floral parts and pollens (Grace and Logan, 2000). In vitro, tocopherol and
ascorbic acid are less effective antioxidant than polyphenols. Polyphenols
contain an aromatic ring with OH or OCH3 substituents which together
responsible for their biological activity. Polyphenols have stronger capacity to
donate electrons or hydrogen atoms because of this; they are powerful
antioxidant in in-vitro. Flavonoids are the major component of polyphenols
and they are usually accumulates in the plant vacuole as glycosides but they
also present in the form of exudates on the surface of leaves and other aerial
parts. Flavonoids can be categorized according to their structure into







environmental conditions, flavonoids serves as a ROS scavenger by locating

and neutralizing radicals before they cause damage to cells (Wang et al.,
2010). Mutants unable to accumulate flavonoids or phenolic compounds were
found to be more sensitive to UV light (Filkowski et al., 2004). Flavonoids are
also providing resistance against pathogens and acting as feeding deterrents.
6. Proline:- Proline is considered one of the member of non-enzymatic
antioxidants required by plants, microbes and animals to mitigate the harmful
effects of ROS. Being an osmolyte now it is considered as a potent antioxidant
and potential inhibitor of PCD (Gill et al., 2010). There is dramatic
accumulation of proline under various adverse environmental conditions such
as salt, drought and metal stress. This may be due to increased synthesis or

decreased degradation of proline. It provide protection against ROS inducedoxidative damage by acting as an osmoprotectant, a metal chelator, a protein
stabilizer, an inhibitor of lipid peroxidation, and OH . and 1O2 scavenger
(Trovato et al., 2008). Proline is more effective scavenger of ROS mainly OH .
than sorbitol, mannitol, myo-inositol. Proline plays an important role in
maintaining the level of NADPH in cells by potentiating pentose-phosphate
pathway. Through this Proline is able to maintain GSH and ASH in the
reduced state. Proline also accumulate in water stress deficit conditions
(Stewart, 1981) and perform an important function as protective compatible
osmolyte in scavenging free radicals and facilitates a correction of altered
redox potential (Harre et al., 1999).
Enzymatic antioxidants:1. Superoxide dismutase (SOD):- SOD is a metalloenzyme and the most effective
intracellular enzymatic antioxidant that plays central role in defense against oxidative
stress induced by ROS in all aerobic organisms (Gill et al., 2010). This enzyme is
present in most of the subcellular compartments that generate activated oxygen (Chen
et al., 2010). This enzyme removes superoxide radical by dismutation into O 2 and
H2O2 and hence decreases the risk of OH . formation (more toxic than H2O2) through
the metal catalyzed Haber-Weiss reaction. SODs are classified into three main types
depending on their metal cofactors: Cu/Zn SOD is structurally somewhat different
from other two SODs, this isozyme is localized to chloroplasts, peroxisomes and
cytosol; Fe-SOD is localized to chloroplasts and Mn-SOD is localized to
mitochondria (del Rio et al., 1996). Different forms of SOD are nuclear encoded and
by an amino terminal targeting sequence, able to targeted to their respective
subcellular compartment.

There are two different techniques to assess the activity of SOD isozymes: negative
staining and to check sensitivity towards KCN and H 2O2. The Cu/Zn-SOD is sensitive
to both inbibitors but Fe-SOD is resistant to KCN and sensitive to H 2O2 (del Rio et
al., 1998). The prokaryotic Mn-SOD and Fe-SOD, and eukaryotic Cu/Zn-SOD
enzymes are dimers, whereas Mn-SOD of mitochondria is tetramer. There is
upregulation of SODs in combating oxidative damages caused due to various stresses
and have a central role in providing tolerance under stress environment. In Mulberry
plant, there is significant increase in SOD activity under saline condition (Harinasut,
2003). In Arabidopsis, it has been reported that there are three genes for Fe-SOD and
Cu-SOD and one gene for Mn-SOD (Kliebenstein, 1999). The activity of SOD is
directly proportional to the increase tolerance of the plant against environmental
2. Catalase:- Catalase is a tetrameric heme-containing antioxidant enzyme. It was the
first enzyme to be discovered and characterized. This enzyme is ubiquitous in nature
and its function is to catalyze the dismutation of two molecules of H 2O2 to water and
O2/minute, thus it has highest turnover rates (Gill et al., 2010) but a much lower
affinity for H2O2 than ascorbte peroxidase (APX). In plants, peroxisome is the major
site for H2O2 generation during photorespiratory oxidant, -oxidation of fatty acids,
purine catabolism, photorespiration and other enzymes systems such as XOD coupled
to SOD (del Rio et al., 2006). Catalase shows strong activity towards H2O2 but have
weak activity against organic peroxides. Catalase does not require reducing

equivalents for degrading H2O2. Catalase enzyme is highly sensitive to light

(Karunppanapandian, 2011). The three CAT genes are found in all angiosperms.
Willekens et al classified CAT into three types depending on the expression profile of
the tobacco genes (Willekens et al., 1995). Class I CATs are found in photosynthetic
tissues and are regulated by light. Vascular tissues contain high level of Class II CATs
and Class III CATs are abundant in seeds and young seedlings. Over expression of a
CAT genes in a plant result in increased tolerance towards oxidative stress (Guan et
al., 2009).

3. Guaiacol peroxidase (GPOX):- GPOX is a heme-containing monomeric protein of

molecular weight appproximately 40 to 50 KDa. It is widely found in all organisms. It
mostly prefers to oxidize aromatic electron donor such as guaiacol and pyrallol by
consuming H2O2. GPOX has a role in the biosynthesis of lignin, biosynthesis of
ethylene, wound healing and decomposition of indole-3-acetic acid. It also act as a
enzymatic antioxidant by consuming H2O2 in the cytosol, vacuole, and cell wall as
well as in extracellular space and provide defense against biotic stresses
(Karuppanapandian et al., 2011). Structurally, these enzymes have four conserved
disulphide bridges and contain two structural Ca2+ ions (Schuller et al.,1996). GPOXs
are widely accepted as stress enzyme. Under stress conditions, GPOX can function
as effective quencher of reactive intermediary forms of O2 and peroxy radicals. Under
salinity stress, it has been noted that induction of GPOX activity in common bean
(Phaseolus vulgaris) nodules (Jebara et al., 2005).
4. Glutathione reductase (GR):- It is a ubiquitous disulphide flavo-protein
oxidoreductase, found in both eukaryotes and prokaryotes (Puertas et al., 2006). It
occurs mostly as a low molecular weight thiol antioxidant which takes part in

enzymatic as well as non-enzymatic oxidation-reduction cycles, GSH is oxidized to

GSSG. It functions as an antioxidant or reductant to protect the thiol groups of
enzymes, regenerate ascorbate and scavenge various forms of ROS. GR catalyzes the
rate limiting last step of the Halliwell-Asada pathway. It is a NAD(P)H-dependent
enzyme involved in the reduction of GSSG to GSH and is essential for maintaining
the GSH pool (Reddy et al., 2006). GR and GSH play a key role in determing the
tolerance of a plant under various abiotic and biotic stresses (Chalapathi et al., 2008).
Around 80% of GR activity is found in photosynthetic tissues and although various
isoforms of GR are also located in chloroplast, cytosol, mitochondria and
peroxisomes (Edwards et al., 1990). In chloroplasts, H2O2 (produce due to Mehler
reaction) detoxification can take place by GSH and GR. If there is increase in GR
activity ultimately results in increase of GSH which confers stress tolerance in plants.

Fig. Asada-Halliwell pathway of hydrogen peroxide scavenging and ascorbic acid

regeneration involving various antioxidant enzymes (Ajay Arora, R.K. Sairam,
G.C.Srivastava, 2002).
5. Monodehydroascorbate reductase (MDHAR):- In the regeneration of reduced
ascorbate, two enzymes are involved, one of them is MDHAR. MDHAR is a
NAD(P)H-dependent enzyme that is present as chloroplastic and cytosolic isozymes.

It catalyzes the regeneration of ascorbic acid from MDHA using NAD(P)H as the
electron donor (Hossain et al., 1985). MDHAR is the only enzyme which use organic
radical as a substrate (MDHA) and also able to reduce phenoxy radicals which are
generated by horseradish peroxidase with H2O2 (Sakihama et al., 2000). This enzyme
is widespread in plants. It also involves in the photoreduction of dioxygen to O 2.

when the substrate MDHA is absent (Miyake et al., 1998). It has been noted that the
overexpression of MDHAR in transgenic tobacco increased tolerance against salt and
osmotic stresses (Eltayeb et al., 2007).
6. Dehydroascorbate reductase (DHAR):- DHAR is the second enzyme involved in
the regeneration of reduced ascorbate from oxidized state (DHA) using GSH as the
reducing substrate (Ushimaru et al., 1997). Reduced ascorbate is major antioxidant in
plants that detoxify ROS, provide tolerance and maintains photosynthetic function
under various stress conditions. DHAR is an important regulator of ascorbic acid
recycling. Overexpression of DHAR activity results in increased tolerance to various
7. Ascorbate peroxidase (APX):- This enzyme is involved in the scavenging of ROS
(H2O2) from chloroplasts and cytosol of plant cells. It is a central component of ASHGSH cycles and also involved in H2O2 scavenging in water-water cycle, and plays an
essential role in the control of intracellular ROS levels. This enzyme utilizes two
molecules of ascorbate as hydrogen donor to reduce H 2O2 to water and production of
two molecules of MDHA. The APX family consists of at least 5 different isoforms
including thylakoid, glyoxisome, membrane as well as chloroplast stromal and
cytosolic form (Noctor and Foyer, 1998). The cytosolic form of APX are two in
number which have several functions like H2O2

scavenging, defensive role and

control electron transport in conjugation with the ascorbate-glutathione cycle (Foyer


et al., 2005). It is more efficient in H2O2 scavenging than catalase and peroxidase and
it may have a key role in the maintained ROS level during stress. It is a member of
class I super family of heme peroxidase (Welinder, 1992) and regulated by redox
signals and H2O2 (Patterson et al.,1995). Many coworkers have reported enhanced
activity of APX in response to various stresses.
8. Glutathione-S-transferases (GST):- It catalyses the conjugated product of
electrophilic xenobiotic substrates with the tripeptide glutathione (GSH: -glu-cysgly). The plant glutathione transferases, formerly known as glutathione-S-tansferases.
It plays a major role in the detoxification of herbicides and hydroxyperoxide,
hormone sequestration, metabolism of tyrosine, anthocyanin sequestration in vacuole,
apoptosis regulation and in plant responses to various biotic and abiotic stresses
(Dixon et al., 2010). It protects DNA, RNA and proteins from oxidative damages by
removing cytotoxic or genotoxic compounds. GST with the help of GSH can also
reduce peroxides and produce scavengers of cytotoxic and genotoxic compounds.
This enzyme generally found in cytoplasm but various isoforms has also been
reported from microsomal, plastidic, nuclear and apoplastic (Frova, 2003). It has also
been found that GST overexpression also enhance plant tolerance to various abiotic
9. Glutathione peroxidase (GPX):- GPX use tripeptide glutathione (GSH) to reduce
H2O2, organic and lipid hydroperoxides, and therefore defense plant cells from
oxidative stress (Noctor et al., 2002).



ROS are produced in both stressed and non-stressed conditions in plants; however, under
adverse conditions, the equilibrium between production and their scavenging is disturbed.
When the level of ROS exceeds the antioxidants, a cell is said to be in a state of Oxidative
stress. Under favourable growth condition, ROS production is low in different cellular
compartment which is produced as by-products of various metabolic pathways. Various biotic
and abiotic stresses lead to the enhanced production of ROS in plants which are destructive in
nature and cause degradation of pigments, carbohydrates, proteins, lipids, nucleic acids,
inactivation of enzymes, destruction of membranes and vital cellular organelles in plants
which ultimately result in cell death. Under stress conditions, several forms of ROS are
produced such as free radical (superoxide radical; hydroxyl radical; perhydroxy radical and
alkoxy radical) and molecular forms include hydrogen peroxide and singlet oxygen. In
addition to destructive macromolecules at high concentrations, ROS also act as a diffusible
signalling molecule at low concentration that perform role in signal transduction pathways as
well as act as a secondary messenger in various developmental pathways and mediate several
plant responses under stress conditions. To mitigate the harmful effects of ROS, plants
possesscomplex antioxidative defence systems. These defence systems comprising both
enzymatic and non-enzymatic components which are involved in the efficient scavenging of
excess ROS produced during various stresses conditions.


A. Arora, R. K. Sairam and G.C. Srivastava, Oxidative stress and antioxidative system in plants,
Current Science, vol. 82, no. 10, pp. 1227-1238, 2002.
A. Cona, G. Rea, R. Angelini, R. Federico, and P. Tavladoraki, Functions of amine oxidases in plant
development and defence, Trends in Plant Science, vol. 11, no. 2, pp. 8088, 2006.










B. Halliwell, Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic
life, Plant Physiology, vol. 141, no. 2, pp. 312322, 2006.
B. Meriga, B. K. Reddy, K. R. Rao, L. A. Reddy, and P. B. K. Kishor, Aluminium-induced production
of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa), Journal
of Plant Physiology, vol. 161, no. 1, pp. 6368, 2004.
C. H. Foyer and G. Noctor, Redox regulation in photosynthetic organisms: signaling, acclimation,
and practical implications, Antioxid Redox Signal,vol. 11:pp. 861905, 2009.
C. H. Foyer and G. Noctor, Oxygen processing in photosynthesis: regulation and signalling, New
Phytologist, vol. 146, no. 3, pp. 359388, 2000.
C. H. Pang and B. S. Wang, Oxidative stress and salt tolerance in plants, in Progress in Botany, U.
Luttge, W. Beyschlag, and J.Murata, Eds., pp. 231245, Springer, Berlin, Germany, 2008.
C. Xiang, B.L. Werner, E.M. Christensen, D.J. Oliver, The biological functions of glutathione
revisited in Arabidopsis transgenic plants with altered glutathione levels, Plant Physiol., vol. 126, pp.
564-574, 2001.
E. F. Elstner, Mechanisms of oxygen activation in different compartments of plant cells, in Active
Oxygen/Oxidative Stress and Plant Metabolism, E. J. Pell and K. L. Steffen, Eds., pp. 1325,
American Society of Plant Physiologists, Rockville, Md, USA, 1991.
E. Lopez-Huertas, F. J. Corpas, L. M. Sandalio, L. A. del Ro, Characterization of membrane
polypeptides from pea leaf peroxisomes involved in superoxide radical generation, Journal of
Biochemistry, vol. 337, pp. 531536, 1999.
Fridovich, I., Annu. Rev. Biochem., vol. 65 pp.97-112, 1995.
G. Noctor and C. H. Foyer, Ascorbate and glutathione: keeping active oxygen under control, Annual
Review of Plant Biology, vol. 49, pp. 249279, 1998.
G. Noctor, S. Veljovic-Jovanovic, S. Driscoll, L. Novitskaya,C. H. Foyer, Drought and oxidative
load in the leaves of C3 plants: a predominant role for photorespiration? Ann. Bot., vol. 89, pp. 841
850, 2002.
G. Noctor, L. Gomez, H. Vanacker, C.H. Foyer, Interactions between biosynthesis,
compartmentation, and transport in the control of glutathione homeostasis and signalling, J. Exp.
Bot., vol. 53, pp. 1283-1304, 2002.
I.M. Moller, P.E. Jensen, A. Hansson, Oxidative modifications to cellular components in plants,
Annu. Rev. Plant Biol., vol. 58, pp. 459-481, 2007.
J. A. Hernndez, M. A. Ferrer, A. Jimnez,A. R. Barcel, F. Sevilla, Antioxidant systems and
O2/H2O2 production in the apoplast of pea leaves. Its relation with salt-induced necrotic lesions in
minor veins, Plant Physiol., vol. 127, pp. 817831, 2001.
K. Asada, Production and scavenging of reactive oxygen species in chloroplasts and their functions,
Plant Physiology, vol. 141, no. 2, pp. 391396, 2006.
K. Asada, Radical production and scavenging in the chloroplasts, in Photosynthesis and the
Environment, N. R. Baker, Ed., pp. 123150, Kluwer, Dordrecht, The Netherlands, 1996.
K. Asada, The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of
excess photons, Annual Review of Plant Biology, vol. 50, pp. 601639, 1999.
K. J. Davies, Protein damage and degradation by oxygen radicals. I. general aspects, Journal of
Biological Chemistry, vol. 262, no. 20, pp. 98959901, 1987
L.A. del Ro, F.J. Corpas, L.M. Sandalio, J.M. Palma, M. Gmez, J.B. Barroso, Reactive oxygen
species, antioxidant systems and nitric oxide in peroxisomes, J. Exp. Bot., vol. 53, pp. 1255-1272,
L-O Klotz, Oxidant-induced signaling: effects of peroxynitrite and singlet oxygen, Biol. Chem.,
vol.383, pp. 44356, 2002.
M. Valko, C.J. Rhodes, J. Moncol, M. Izakovic, M. Mazur, Free radicals, metals and antioxidants in
oxidative stress-induced cancer, Chem. Biol. Interac., vol. 160, pp. 1-40, 2006.
M. A. Torres, J.L. Dangl, J. D. G. Jones, Arabidopsis gp91phox homologues AtrbohD and AtrbohF
are required for accumulation of reactive oxygen intermediates in the plant defense response, Proc.
Natl. Acad . Sci ., USA , vol. 99, pp. 517522 , 2002.
N. Navrot, N. Rouhier, E. Gelhaye, J.P. Jaquot, Reactive oxygen species generation and antioxidant
systems in plant mitochondria, Plant Physiology, vol. 129, pp. 185-195, 2007.


26. N. Smirnoff, Ascorbate, tocopherol and carotenoids: metabolism, pathway engineering and functions.
in: N. Smirnoff (Ed.), Antioxidants and Reactive Oxygen Species in Plants, Blackwell Publishing
Ltd., Oxford, UK, pp. 53-86, 2005.
27. P. Sharma, A. Bhushan Jha, R. Shankar Dubey, and M. Pessarakli, Reactive oxygen species, oxidative
damage, and antioxidative mechanisms in plants under stressful conditions Journal of Botany, pp. 126, 2012.
28. P. Sharma, A. B. Jha, and R. S. Dubey, Oxidative stress and antioxidative defense system in plants
growing under abiotic Stresses, in Handbook of Plant and Crop Stress,M. Pessarakli, Ed., pp. 89
138, CRC Press, Taylor and Francis Publishing Company, Fla, USA, 3rd edition, 2010.
29. P. Ghezzi, V. Bonetto, Redox proteomics: identification of oxidatively modified proteins,
Proteomics, vol. 3, pp. 1145-1153, 2003.
30. Prasad PVVPisipati SR, Momilovi I, Ristic Z. Independent and combined effects of high
temperature and drought stress during grain filling on plant yield and chloroplast EF-Tu Expression in
spring wheat,.Journal of Agronomy Crop Science, vol. 197, pp.430-441, 2011
31. R. Mittler, Oxidative stress, antioxidants and stress tolerance, Trends Plant Sci., vol. 7, pp. 405-410,
32. R. Mittler , S. Vanderauwera, M. Gollery, F. Van Breusegem, Reactive oxygen gene network of
plants, Trends Plant Sci., vol. 9, pp. 490498, 2004.
33. S. Bhattacharjee, The language of reactive oxygen species signaling in plants, Journal of Botany, 1-22, 2012.
34. S. Bhattachrjee, Reactive oxygen species and oxidative burst: roles in stress, senescence and signal
transduction in plant,Current Sci., vol. 89, pp. 1113-1121, 2005.
35. S. Singh Gill and N. Tuteja, Reactive oxygen species and antioxidant machinery in abiotic stress
tolerance in crop plants, Plant Physiology and Biochemistry, vol. 48, pp. 909-930, 2010.
36. S. Kukreja, A.S. Nandval, N. Kumar, S.K. Sharma, S.K. Sharma, V. Unvi, P.K. Sharma, Plant water
status, H2O2 scavenging enzymes, ethylene evolution and membrane integrity of Cicer arientinum
roots as affected by salinity, Biol. Plant., vol. 49, pp. 305-308,2005.
37. S. Hatz, J. D. C. Lambert, and P. R. Ogilby, Measuring the lifetime of singlet oxygen in a single cell:
addressing the issue of cell viability, Photochemical and Photobiological Sciences, vol. 6, no. 10, pp.
11061116, 2007.
38. S. Hackbarth, J. Schlothauer, A. Preu, and B. Roder, New insights to primary photodynamic effects
singlet oxygen kinetics in living cells, Journal of Photochemistry and Photobiology B, vol. 98, no.
3, pp. 173179, 2010.
39. S. Mishra, A. B. Jha, and R. S. Dubey, Arsenite treatment induces oxidative stress, upregulates
antioxidant system and causes phytochelatin synthesis in rice seedlings, Protoplasma, vol. 248, no. 3,
pp. 565577, 2011.
40. T. Liu, J. Van Staden, and W. A. Cress, Salinity induced nuclear and DNA degradation in
meristematic cells of soybean (Glycine max (L.)) roots, Plant Growth Regulation, vol. 30, no. 1, pp.
4954, 2000.
41. T. Karuppanapandian, J-Cheol Moon, C. Kim, K. Manoharan, W. Kim, Reactive oxygen species in
plants: their generation, signal transduction, and scavenging mechanisms, Australian Journal of Crop
Science, vol. 5, no. 6, pp. 709-725, 2011.
42. Tan, W, Meng, Q. W, Brestic, M, Olsovska, K, & Yang, X., Photosynthesis is improved by exogenous
calcium in heat-stressed tobacco plants, Journal of Plant Physiology, vol.168, pp. 2063-2071, 2011.