Abiotic stress in plants: mechanism and management strategies

Submitted To DR. M Balakrishnan

Submitted By

Shashi Meena Scientist- trainee FOCARS-101 st

ICAR-National Academy of Agricultural Research Management Rajendranagar, Hyderabad-500030, Telangana, India

Index 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 Drought Salinity Chilling Metal toxicity High temperature ROS scavenging mechanism in plants------------------------------------------------19-20Non-enzymatic antioxidants----------------------------------------------------------20-28 Ascorbic acid (Vitamin-C) Glutathione (GSH) Tocopherol (Vitamins-E) Carotenoid Phenolic compounds Proline 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) Conclusion------------------------------------------------------------------------------------34 References-----------------------------------------------------------------------------------35-37 Abiotic stress in plants: mechanism and management strategiesIntroduction:-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.

Sources of ROS:-

MITOCHONDRIACHLOROPLAST

PLASMA MEMBRANE

CELL WALLROS

APLOPLAST

ENDOPLASMIC RETICULUM

PEROXISOME

GLYOXYSOME Fig: Main sources of ROS in plant cellGround 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 O2, 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 O2 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 (O2.-), 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 O2. O2.- can also react with another signalling free radical species, NO. give up peroxynitrate (OONO-) (Gill et al.,2010).Dioxygen e- Superoxide radical e- peroxide e- O23- Oxene O2- (3O2) ion (O2.-) ion (O22-) ion (O-)

WaterHydroxyl radical (OH.)Water (H2O)Hydrogen peroxide (H2O2)Perhydroxyl radical (HO2.)Singlet oxygen (1O2)

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 O2. 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 H2O2. This process is carried out either spontaneously or by superoxide dismutase (SOD). Furthermore, O2.- can also donate electron to Fe3+ to yield a reduced form of iron (Fe2+). 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, H2O2 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 (-SCH3), 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., 2008). 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., 2002).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).

ANTIOXIDANTS Fig: The graph showing relationship between

STRESS CONDITIONS antioxidants and stress.

ABIOTIC STRESSLipid peroxidation ROS

Protein oxidation Oxidative damage

Alter membrane propertiesEnzyme inactivation

DNA damageDamage to photosynthetic machinery

CELL DEATH

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) (Lipid Alkyl radical) Propagation:- The peroxy radical formed is highly reactive and able to propagate the chain reaction.Propagation steps:-R. + O2 ROO. (Lipid peroxy radical)ROO. + RH ROOH + R.ROOH 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. R + R (Fatty acid dimer)R. + ROO. ROOR (Peroxide bridged dimer)ROO. + ROO. ROOR + O2 (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 three-dimensional 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-2-deoxyguanosine 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 O2 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 CO2 assimilation (Noctor et al., 2002). Sangmein lee and Chung-MO Park found that the NTL4 gene is induced in the response of H2O2, 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 H2O2 as a consequence of increase in its oxygenase reaction. Under HT condition, the stomatal conductancealso decreased significantly. Prasad et al. reported that high night temperature (31.9C/27.8C) decreased chlorophyll (Chl) content and 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), Guaiacol peroxidase (GPOX), Superoxide dismutase (SOD), Monodehydroascorbate redutase (MDHAR), Dehydroascorbate reductase (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., 2001).

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 H2O2 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., 1997).

Dehydroascorbate reductaseDehydroascorbate +Glutathione (GSH) Ascorbic acid + Glutathione (GSSG)

Glutathione reductaseGlutathione (GSSG) + NADPH 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 O2 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 diverse secondary metabolites including flavonoids, tannins, 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 flavonols, flavones, isoflavones and anthocyanin. Under adverse 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 induced- oxidative 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 O2 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 H2O2. The Cu/Zn-SOD is sensitive to both inbibitors but Fe-SOD is resistant to KCN and sensitive to H2O2 (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 stresses.

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 H2O2 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 O2. - 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 stresses.

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 ASH-GSH 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 H2O2 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-cys-gly). 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 stresses.

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).

Conclusion:-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.

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