Abstract
Environmental pollution caused by heavy metals has received worldwide attention. The intractable and pertinacious nature of heavy metals leads to severe threat to environment, and affects the life of both plants and animals, causing serious diseases in humans. Heavy metal toxicity in plants cause imbalances in the redox metabolism that leads to oxidative damage which is characterized by enhanced production of reactive oxygen species (ROS). To minimize the deleterious consequences of ROS, plants in general have developed biological detoxification and defense mechanisms that protect the cellular components from being oxidized. Antioxidative defense activity of plants is composed of enzymatic scavengers such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and guaiacol peroxidase (GPX) and non-enzymatic components like ascorbic acid (AA), α-tocopherol, carotenoids, flavonoids and proline that plays the most crucial and effective role in detoxifying ROS and the changes in their activity is often used to predict metal tolerance. In this chapter, the current state of knowledge about the role of ROS, defense mechanisms and adaptation strategies of plants with special reference to antioxidative defense system to alleviate heavy metal toxicity is discussed. Recent researches have thrown ample lights on how enzymatic and non-enzymatic machinery of plants can protect, regulate and integrate cell responses to heavy metal stress.
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Keywords
- Antioxidant defense
- Oxidative stress
- Reactive oxygen species
- Secondary metabolism
- Metal chelation
- Glutathione
- Phytoremediation
3.1 Introduction
Early on, metal pollution only affected the small portion of the population which was in close proximity of the metal source. However, at the turn of the nineteenth century, the start of the industrial revolution resulted in a greater demand for products such as coal, iron and steel. Pollutants were no longer restricted to small areas, but instead, were distributed over a wide area, by means of air and water. This has caused deleterious effects to the ecosystem and human health. Today, many soils throughout the world have been known to be polluted with heavy metals.
Heavy metals include arsenic (As), silver (Ag), lead (Pb), cadmium (Cd), zinc (Zn), mercury (Hg), chromium (Cr), copper (Cu) iron (Fe), etc. Heavy metals are better described as ‘trace metals’—metals found at low concentrations, usually less than one ppm or less, in a specified source, e.g. soil, plant tissue or groundwater (Phillips 1981). Heavy metals are not considered as a pollutant if persist in low concentrations. They occur naturally in the environment due to their presence in bedrocks. Some heavy metals such as zinc and copper are also essential micronutrients for living organisms. The term ‘heavy metal pollution’ refers to heavy metal levels that are relatively higher than the normal background concentration; mere presence of the metal is insufficient evidence of pollution (Alloway and Ayres 1997).
Heavy metal concentrations in soil range from less than 1 mg kg−1 to over 1000 mg kg−1 (Adriano 2003). They are present in the earth’s crust naturally in different minerals at different concentration and many of these metals are essential for cells (e.g. Cu, Fe, Mn, and Zn) (Marschner 1995). The world-wide emissions of metals to the atmosphere by natural sources is estimated as: Ni: 26, Pb: 19, Cu: 19, As: 7.8, Zn: 4, Cd: 1.0, Se: 0.4, (thousand tonnes year−1). Whereas, from anthropogenic sources: Pb: 450, Zn: 320, Ni: 47, Cu: 56, As: 24, Cd: 7.5, Se: 1.1 (thousand tonnes year−1) (Valavanidis and Vlachogianni 2010). It is obvious from these numbers that Pb, Zn, Ni and Cu are the most important metal pollutants generated by anthropogenic activities.
3.2 Heavy Metals and Their Impact on Plants
3.2.1 Cadmium
Cadmium is commercially used in paint pigments, glass making, electroplating, photography, dying, stabilizer, and in batteries. It is classified as a probable human carcinogen (group B1) by the USEPA and toxic to organisms. In the past, there have been examples of marked Cd contamination in areas where food has been grown. This was particularly so for rice crops in Japan where Cd concentrations from 200 to 2000 ppb were found (Elinder 1985). In general, soils which have been historically contaminated with Cd from industrial operations are now no longer used for agricultural purposes. The accumulation of Cd in water and soil has caused major environmental and human health concern. It is a biologically non-essential metal, toxic to both plant and human beings (Shukla et al. 2007).
Plants upon exposure to Cd show various symptoms such as chlorosis, growth inhibition, burning of root tips, and finally death (Kahle 1993). Cd affects stomatal function, water transport and cell wall elasticity (Baszynskiet al. 1980). Baryla et al. (2001) reported an increase in the stomatal resistance of plants that were treated with Cd. Inhibition of photosynthesis is another toxic effect of Cd, which is brought about by reduced stomatal conductance in response to metal toxicity and sensitivity of photosystem II to high Cd concentration. Cd may affect PS II on both the oxidising (donor) and reducing (acceptor) side (Haag-Kerwer et al. 1999). Rubisco activity in the Calvin cycle is inhibited by high Cd. The clearest symptom of Cd phytotoxicity is leaf chlorosis. High Cd concentration in the plant induces increased respiration and activities of the tricarboxylic acid cycle as well as other pathways of carbohydrate utilisation (Arisi et al. 2000).
Hyperaccumulation of Cd is a rare phenomenon in higher plants. Thlaspi caerulescens has been identified as Cd hyperaccumulator (Baker et al. 2000). Ecotypes of T. caerulescens accumulate up to 12,500 mg kg−1 Cd dry weight, without showing signs of toxicity (Peer et al. 2003). Cd hyperaccumulation in Arabidopsis hallerii has also been reported (Cosio et al. 2004). Vetiver grass could be used to remediate Cd-polluted soil, since it accumulated 218 g Cd ha−1 at a soil Cd concentration of 0.33 mg Cd kg−1 (Chen et al. 2000). Several plant species have been identified which are capable of accumulating and tolerating moderate to high levels of Cd in their tissues such as Solanum lycopersicum, Chenopodium murale, Poa annua, Calotropis procera, and Abutilon indicum (Varun et al. 2012, 2015; D’Souza et al. 2013).
3.2.2 Arsenic
Arsenic (As) is a naturally occurring metalloid, commonly used in pesticides and wood preservatives. In India, As contamination of groundwater has resulted in widespread well contamination and possess serious health risk. Frequent use of As contaminated water in irrigation resulting in As poisoning of humans and other animals through dietary intake (McArthur et al. 2001). Similar contamination is also been reported from various regions with As in subsoils worldwide.
Arsenite [AsO2 − or As(III)] and arsenate [AsO4 −3 or As(V)] are the dominant inorganic arsenic moieties found in terrestrial plants and both forms are phytotoxic. Arsenate is predominantly found in aerobic soils. Formation of ADP-As complexes instead of ATP leads to cell death. Arsenate is often designated as more phytotoxic of the two arsenic from but the relative toxicities are species-specific (Wang et al. 2002).
Non-accumulators have a phytotoxic threshold at approximately 5–100 mg kg−1As dry weight (wt.). Fern pteriodophyte, Pteris vittata can accumulate As in contaminated as well as non-contaminated soils suggesting that hyperaccumulation is a constitutive trait (Wang et al. 2002). Holcus lanatus can accumulate upto 560 mg kg−1 As, and Pteris vittata can accumulate up to 27,000 mg kg−1 As dry wt., with phytotoxic symptoms appearing around 10,000 mg kg−1As dry wt. (Gumaelius et al. 2004). Ferns like Pteris cretica, P. longifolia, and P. umbrosa can hyperaccumulate As (Wei and Chen 2006). Another fern, Pityrogramma calomelanos has also been discovered as an As hyperaccumulator, it showed great potential in phytoremediation of As contaminated soils (Francesconi et al. 2002).
3.2.3 Mercury
Over the past few decades, several thousand tons of Hg have been released to the environment by human activity (Ebinghaus et al. 1999). Once released into the atmosphere it can be exchanged between soil and water by wind erosion, degassing of mineralized rock formations, and volcanic eruptions. Use of elemental Hg-Au amalgamation in mining is a common practice, which results in significant Hg contamination in the surrounding environment. Organomercurials like methyl-Hg and phenylmercuric acetate are the most toxic forms of Hg followed by ionic Hg(II), while elemental Hg(0) is the least toxic form.
Mercury is a persistent environmental pollutant with bioaccumulation ability in fish, animals, and human beings (Chang et al. 2009). The mechanism and extent of Hg toxicity depend strongly on the type of compound and the redox state of mercury. Terrestrial plants are generally insensitive to the harmful effects of Hg. However, it is known to affect photosynthesis and oxidative metabolism by interfering with electron transport in chloroplasts and mitochondria. Mercury also inhibits the activity of aquaporins and reduces plant water uptake (Sas-Nowosielska et al. 2008).
Hg is toxic to plants, and to date Hg hyperaccumulating plants have not been identified. However, Hg hyperaccumulating Amanita muscaria (mushroom) has been identified and can accumulate 96–1900 ng g−1 dry wt. (Falandysz et al. 2003). Most of the phytoremediation studies focused on converting the organomercurials to Hg(0), which is volatile and is released into the atmosphere. Transgenic poplar and cottonwood trees expressing merA and/or merB could be used as phytoremediators which do not require harvesting or replanting each season (Che et al. 2003). Plants growing on Hg-polluted soils show a reduced growth and vitality (Tomiyasu et al. 2017). Specific Hg-adapted metallophytes do not seem to exist. However, 13 plant species accumulated Hg out of 87 examined from a highly contaminated waste area originating from a chemical plant in Italy (Massa et al. 2010). Polygonum aviculare was identified as the best accumulator; however, its slow growth and low productivity prevents it to be used for phytoextraction purposes.
3.2.4 Chromium
Chromium (Cr) with its great economic importance in industrial world is a major metal pollutant of the environment. It is used in industries for chrome plating, wood preservation, textile dyeing and pigmentation, manufacturing pulp and paper, and tanning (Sharma and Adholeya 2011). Leather tanning industries produce large amounts of organic and chemical pollutants, especially, chromium. This Cr containing untreated effluent and sludge discharged by tanneries possess a serious threat to the environment and human health. Chromium is present in many oxidation states [Cr(II) to Cr(VI)], the most common forms are Cr(0, III, and VI). World Health Agency and EPA has determined Cr(IV) as a carcinogen. It is considered to be 1000 times more toxic than Cr(III). Cr(VI) contamination in the soil and groundwater has been reported in various regions of California, USA (EPA 2004).
Cr is a toxic and non-essential element to plants. The toxic effects of Cr are primarily dependent on the metal speciation, which determines its uptake, translocation and accumulation (Shankar et al. 2005). The two stable forms of Cr i.e. Cr(III) and Cr(VI) are taken up by the plants by different mechanisms. Barcelo et al. (1986) reported an inhibition of micronutrients like P, K, Zn, Cu and Fe translocation within bean plant parts was observed when treated with Cr in growth matrix. Sujatha and Gupta (1996) reported that tannery effluent irrigation caused micronutrient deficiencies in several agricultural crops. Cr induced chlorosis was also observed in rye grass (Ottabbong 1989). Cr compounds are highly toxic to plants and are detrimental to their growth and development (Shankar et al. 2005).
Davies et al. (2002) reported that while some plants are not affected by low Cr concentration (3.8 × 10−4 μM Kg−1), however it is toxic to most of the higher plants at 100 μM Kg−1 dry weight. Zhu et al. (1999) reported that Eichhornia crassipes was a good accumulator of Cr and Cd. Pulford et al. (2001) investigated Zn and Cr content in different tree species and found Cr to be accumulated mainly in roots, whereas Zn was translocated to shoots. Zavoda et al. (2001) found that Helianthus annuus and Brassica juncea accumulates significant Cr in roots.
3.2.5 Copper
Copper (Cu) is not poisonous in its metallic state but some of its salts are poisonous. Cu is an essential element and enzyme co-factor for oxidases (cytochrome c oxidase, superoxide dismutase) and tyrosinases. Cu contamination in soil usually results from mining, pesticides, and industrial wastes. It deposits in the liver and disrupts the liver’s activity thus adversely affect the nervous system, reproductive system, adrenal function, development of the child, etc. (Badiye et al. 2013).
Cu contributes to several physiological processes in plants including photosynthesis, respiration, carbohydrate distribution, nitrogen and cell wall metabolism, seed production and disease resistance (Kabata-Pendias 2001). Leaf chlorosis is an initial symptom of Cu toxicity (Zhu and Alva 1993). Mitchell et al. (1988) reported leaf chlorosis in Banksia ericifolia, Casuarina distyla and Eucalyptus eximia grown at elevated Cu. At higher Cu toxicity, leaves become wilted before eventually becoming necrotic. Copper toxicity can be associated with a purpling of foliage but this is not apparent in all species (O’Sullivan et al. 1997).
Copper toxicity has a significant effect on root-shoot growth and biomass of the plants (Minnich et al. 1987). In a study conducted by Patterson and Olson (1983) the germination of six tree species was found less sensitive to Cu than subsequent root elongation. Citrus paradisi x Poncirus trifoliata seedlings exposed to excess Cu produce few new roots and have a thickened tap root (Zhu and Alva 1993). Most Cu-tolerant plants are excluders, and no confirmed Cu accumulators have been identified to date. Liao and Chang (2004) identified an aquatic weed; Eichhornia crassipes can accumulate 21.62 kg Cu ha−1, and could be used for phytoremediation Cu contaminated waste water. Elsholtzia haichowensis has been identified as a Cu tolerant and accumulating plant species in mining areas (Yang et al. 2002).
3.2.6 Zinc
Zinc (Zn) is considered as one of the most important micronutrients for animals and plants but on the other hand is toxic if it exceeds the sufficient level (Hambidge and Krebs 2007). About 70% of the world’s Zn originates from mining, while the remaining 30% comes from recycling secondary zinc. Farm manures also have high concentrations of Zn, which make them a promising amendment for Zn-deficient soils. Zinc is an essential element for living organisms and is toxic to living organisms only at very high concentrations (McIntyre 2003). It is an essential component for protein production in all terrestrial life and is the second metal only to iron in terms of abundance in living organisms.
The first symptom of Zn toxicity exhibited by most plants is a general chlorosis of the younger leaves (Fontes and Cox 1995). This chlorosis can further progress to reddening due to anthocyanin production in younger leaves. Plants exhibiting Zn toxicity have smaller leaves than their control plants (Ren et al. 1993). Zinc stressed Glycine max exhibit vertically oriented leaves (Fontes and Cox 1995). In severe cases, plants may exhibit necrotic lesions on leaves and eventually entire leaf death. In roots, Zn toxicity is apparent as a reduction in the growth of the main root, fewer and shorter lateral roots and a yellowing of roots (Ren et al. 1993). Khurana and Chatterjee (2001) reported a reduction in biomass, seed number, seed weight and soluble proteins in sunflower (Helianthus annuus) plants grown in Zn-laden soil.
The first Zn hyperaccumulator identified was Thlaspi caerulescens. It can accumulate 25,000–30,000 μg g−1 Zn, although T. caerulescens can accumulate a maximum dry weight of 40,000 ug g−1 Zn in its shoots (Pence et al. 2000). Arabidopsis halleri has also been found to increase in its shoot Zn concentration from 300 ug g−1 dry wt at 1 μM Zn to 32,000 ug g−1 at 1000 μM Zn without phytotoxicity (Zhao et al. 2000).
3.2.7 Lead
Lead (Pb) has been commonly used for thousands of years because of its widespread occurrence, easy to extract and easy to work with. Pb is the most significant toxin of the heavy metals. It accumulates primarily in the surface soil layer, and its concentration decreases with the soil depth. It may exist in the atmosphere as dust, fumes, mist and vapours and in soil as mineral. Soils along roadsides are particularly lead rich because of pollution from vehicles burning leaded gasoline (Eick et al. 1999). Important sources of lead pollution are geological weathering, industrial processing of ore and minerals, leaching of metals from solid wastes. Once it is introduced into the growth matrix, it is very difficult to remove Pb.
A high Pb level in soil induces abnormal morphology in many plant species. It causes irregular radial thickening in pea roots, cell wall of the endodermis, and lignifications of cortical parenchyma (Paivoke 1983). Lead also induces proliferation effects in the repair process of vascular plants. A significant amount of lead in the plant causes reduction in plant growth (Salim et al. 1995). Lead toxicity can cause plasma membrane alteration in plants because Pb2+ is physiologically similar to Ca2+ (Srivastava and Gupta 1996). Elevated Pb interferes with chlorophyll formation and the normal metabolism of Fe (Kacabova and Natr 1986). High concentration of Pb has been linked to poor seed germination, high stomatal resistance, inhibited CO2 uptake and low photosynthetic rate (Poskuta et al. 1987). Lead induced inhibition of seed germination has been reported in Hordeum vulgare, Elsholtzia argyi, Spartina alterniflora, Pinus halepenis, Oryza sativa and Zea mays (Islam et al. 2007; Senger et al. 2009).
Certain plants (mostly, belonging to the Brassicaceae, Euphorbiaceae, Asteraceae, Lamiaceae, and Scrophulariaceae families) have been identified which have the potential to uptake Pb (USEPA 2000). Many plants have a strategy of Pb exclusion as Thlaspi praecox, which hyperaccumulates Cd and Zn but exclude Pb (Vogel-Mikus et al. 2005), however, several plant species can hyperaccumulate Pb. Sesbania drummondii, Brassica species and Pelarogonium can accumulate significant Pb content in roots (Blaylock et al. 1997; Sahi et al. 2002), without showing any symptoms of toxicity.
3.2.8 Nickel
Nickel (Ni) is an essential element that can be toxic and possibly carcinogenic in high concentrations only. It is found in different concentrations in all soil types of diverse climatic regions. The normal range of Ni in soil is 2–750 ppm, with a critical soil concentration at 100 ppm (Gardea-Torresdey et al. 2005). Exposure to Ni compounds causes irreversible damage to the nervous system, cardiovascular system, lungs and gastrointestinal tract (Axtell et al. 2003). Nickel has been classified among the essential micronutrients and remains associated with some metallo-enzymes, but Ni is toxic at elevated concentrations in plants (Srivastava et al. 2005).
Toxic symptoms of Ni include yellowing, necrosis, chlorosis, deformed and stunted growth and ROS generation (Halliwell and Gutteridge 1999).
Serpentine and ultramafic soils are naturally occurring regions of high Ni concentrations characterized by unique Ni-tolerant flora. The majority of Ni hyperaccumulators have been collected from these soils. Alyssum lesbiacum and Thlaspi goesingense are both Ni hyperaccumulating plants in the Brassicaceae family. In Alyssum genus alone, 48 different species have been discovered containing between 1000 and 30,000 μg g−1 Ni in leaf (Baker and Brooks 1989; Kerkeb and Kramer 2003). Several Ni phytoextraction using hyperaccumulators has been patented (Chaney et al. 1999).
3.3 Reactive Oxygen Species
Anthropogenic activities and natural tragedies such as pollution, drought, salinity, UV radiation, temperature extremes and heavy metals generate different kinds of stresses that adversely affect the biological systems and cellular metabolic processes such as respiration and photosynthesis which unavoidably lead to the production of reactive oxygen species (ROS). ROS are highly reactive component which includes free radicals such as superoxide anion (O2 •−), hydroxyl radical (OH∙), as well as non-radical molecules like hydrogen peroxide (H2O2) and singlet oxygen (1O2). ROS production takes place by a sequenced reduction of molecular oxygen (O2) under high energy subjection or electron-transfer reactions (del Río et al. 2006; Blokhina and Fagerstedt 2010; Heyno et al. 2011). The O2 molecule with two impaired electrons and same spin quantum number act as a toxic and strong oxidizing agent. This spin orientation makes O2 susceptible to accept electrons one at a time that ultimately leads to the generation of the so-called ROS, which generate oxidative stress on cellular compartments. Several metabolic pathways, localized in different cellular compartments such as chloroplast, mitochondria and peroxisomes also contribute to the ROS production continuously as a byproduct of biochemical reactions (Navrot et al. 2007). Under normal conditions, these oxidants are scavenged by various antioxidative defense mechanisms and maintain a cellular equilibrium (Heyno et al. 2011) but heavy metal load can disturb this balance that eventually leads to a rapid increase in intracellular production of ROS which is significantly destructive for biomolecules and different compartments of the cell (Fig. 3.1). It has been reported that 1–2% of O2 consumption leads to the formation of ROS in plant tissues (Bhattacharjee 2005).
High concentration ROS are immensely harmful for plants and when the level of ROS exceeds the threshold level, a cell is said to be in a state of ‘oxidative stress’. Oxidative stress is essentially a regulated process and the destiny of plant cell is determined by its capacity to maintain equilibrium between the oxidative and antioxidative state. ROS affect many cellular functions by damaging nucleic acids, oxidizing proteins, and causing lipid peroxidation (Foyer and Noctor 2005). Changes in the concentration of ROS determine its function, like at low concentration it acts as a signalling factor that trigger the defense machinery of the cell while at high concentration it becomes detrimental for the cell (Gratao et al. 2005).
3.4 Types of ROS
ROS has been well known for playing a dual role as both harmful and beneficial species which is categorised by its intracellular concentration. The most common ROS include singlet oxygen (1O2), superoxide radical (O2 •−), hydrogen peroxide (H2O2) and hydroxyl ions (OH•). Although, atmospheric oxygen is relatively non-reactive in its ground state as it has two unpaired electrons with parallel spin which makes it paramagnetic and, therefore does not participate in reactions with organic molecules until gets activated (Fig. 3.2), but when the cell is exposed to excess excitation energy reversion of one of its unpaired electron takes place that contribute to the emergence of singlet state with two electrons in different spins. This reaction controls the spin orientation of electrons and thereby 1O2 undergoes divalent reduction with concurrent shifting of two electrons. In the process of ROS formation several intermediates are formed as shown in Fig. 3.3.
Under heavy metal stress, highly reactive singlet oxygen (1O2) can be produced via reaction between oxygen and the chlorophyll triplet state. The chlorophyll triplet state can react with 3O2 to give up the highly reactive 1O2 (Krieger 2005). This formation of photosynthetic singlet oxygen may damage the photosynthetic machinery of plants i.e., PSI and PSII (Skovsen et al. 2005). 1O2, an oxidizing agent can damage the molecules like proteins, pigments, nucleic acids and lipids, and is thought to be the most important species responsible for light induced loss of PSII activity which may trigger cell death (Scandalios 2005). 1O2 can be quenched by β-carotene, α-tocopherol or can react with the D1 protein of photosystem II as a target. Therefore, Singlet oxygen plays an important role in providing protection against photooxidative stress (Halliwell 2006).
The formation of superoxide radical (O2 •−) in chloroplast is mainly formed in the thylakoid localized PSI during non-cyclic electron transport chain (ETC), as well as other cellular compartments. It is generally considered to be the first ROS generated under stressful environment. It causes membrane lipid peroxidation by transformation into OH• and 1O2 which are more reactive and toxic. The O2 •− is synthesised through reduction of O2 during electron transport of chloroplasts and other compartments of the plant cell. O2 •− is a nucleophilic reactant with both oxidizing and reducing properties that can be generated by electrons flow in PS I which accelerate the production of other highly harmful radicals like OH•, and more possibly 1O2 (Halliwell 2006).
During photooxidation reactions, NADPH oxidase as well as xanthine oxidase (XOD) also contributes to H2O2 production in plants. H2O2 is moderately reactive and is relatively long-lived molecule with a half-life of 1 ms (Allen et al. 1997). H2O2 has no unpaired electrons, unlike other oxygen radicals; it can cross membrane via aquaporins and consequently can cause oxidative damage far from the site of its formation. It may inactivate enzyme by oxidizing their thiol group, such as Calvin cycle, Cu/Zn-SOD, and Fe-SOD.
Hydroxyl radical is one of the most dangerous radical as it has a single unpaired electron, thus it can react with oxygen in triplet ground state. Hydroxyl radical has the capability to damage different cellular components such as lipid peroxidation (LPO), protein damage and protein destruction (Foyer et al. 1997). Excess formation of OH˙ can lead to cell death because the cells have no enzymatic mechanism to eliminate OH.
3.5 Sites of Production of ROS
ROS are continuously and predominantly produced at several locations in chloroplasts, mitochondria, peroxisomes, plasma membrane, endoplasmic reticulum and cell wall. But when ROS is formed by the inevitable leakage of electrons and a number of adverse abiotic stress factors such as light, drought, low temperature and heavy metal stress, it may cause significant damage to plants cell structures, whereas at low concentrations, it acts as secondary messenger for the plant.
3.5.1 Mitochondria
Mitochondria is known as “energy factory” and it is a major site for ROS production (Navrot et al. 2007). Plant mitochondria differ from animal counterparts by being involved in photorespiration. The cellular environment of plant mitochondria is also distinctive because of the presence of photosynthesis, which creates O2 and carbohydrate rich environment (Noctor et al. 2006). When plants are exposed to heavy metal toxicity, distribution and transport of metal ions to apoplast followed by cytosolar uptake causes ROS generation due to their redox-active nature (Sharma and Dietz 2009). The Major components of mtETC responsible for ROS production is complex I and complex III. Mitochondrial matrix contains several enzymes which produce ROS but some of them produce ROS directly like aconitase and other like 1-galactono-ɣ-lactone dehydrogenase (GAL) which indirectly feeds electrons to ETC (Rasmusson et al. 2008). O2 •− is the primary ROS formed by monovalent reduction in the ETC. In aqueous solution, O2 •− is moderately reactive, but this O2 •− is converted to H2O2 by the Mn-SOD and the APX (Sharma et al. 2012). Abiotic stresses like heavy metals plays a major role as they affect the plant cell bioenergetics and influence the tight coupling of ETC and ATP generation by reducing the electron carriers like ubiquinone thus ultimately generate oxidants (Blokhina and Fagerstedt 2010). Transition metals such as Cu, Fe and Zn are crucial for a proper functioning of several enzymes involved in the TCA cycle, electron transport, synthesis of ATP and antioxidative defense (Nouet et al. 2011; Tan et al. 2010). Findings of several studies focusing on plant metal stress responses point toward the emergence of oxidative stress and mitochondrial dysfunction as determinants in metal-induced cytotoxicity. In several plant species, metal stress enhances mitochondrial ROS generation mainly by affecting respiratory gas exchange rates (Losch 2004).
3.5.2 Chloroplast
Chloroplast is an important cell organelle which plays an important role as photosynthesis takes place here. It is a highly complex cell organelle which contains organised thylakoid membrane system that harbours all components of the light-capturing photosynthetic apparatus. PSI and PSII are the main sources of ROS in Chloroplasts (Fig. 3.4). ROS production by these sources is enhanced in plants by abiotic factors such as drought, salinity, temperature extremes as well as combination of these conditions with high light stress (Fannschmidt 2003). Normally, the electron flow from the excited centre to PS centre and ultimately NADP which is then reduced to NADPH and enters the Calvin cycle and reduce the final electron acceptor, CO2. Due to decreased NADP supply resulting from stress conditions, there is a leakage of electron from ferredoxin to O2, reducing it to O2 •− (Elstner 1991).
PSI and PSII plays an important role in the production of ROS. The site of leakage of electrons from the ETC of PSI is 2Fe-2S and 4Fe-4S clusters. PSII acceptor side of ETC contains QA and QB which is responsible for the generation of O2 •− (Cleland and Grace 1999). PS II contains a reaction centre P680 from which the electrons get excited. PS II is responsible for the generation of 1O2, which occurs in two ways, firstly, when abiotic stress upsets the delicate balance between light harvesting and energy utilisation which leads to the formation of triplet Chl (3Chl*) which on reacting with dioxygen (3O2) liberates 1O2(Karruppanapandian et al. 2011). Secondly, when ETC is over reduced the light harvesting at PS II to generates 1O2 (Asada 2006) and causes peroxidation of membrane lipids, damage membrane proteins and lead to cell death (Triantaphylids et al. 2008)
3.5.3 Peroxisomes
Peroxisomes are small, dynamic and spherical organelles bounded by a single lipid bilayer membrane and are the major site of intracellular H2O2 production due to integral oxidative metabolism (Palma et al. 2009). The major function of peroxisomes is breakdown of very long chain fatty acids through beta-oxidation. There are two sites for the production of superoxide radical, first site is in the matrix of peroxisomes where xanthine oxidase (XOD) catalyses the oxidation of xanthine and hypoxanthine to uric acid which generates O2 •− and the second one is in the peroxisome membrane dependent on NADPH where a small ETC is composed of NADH and Cyt b and contribute to O2 •− production. Peroxisomes also play an important role in photomorphogenesis degradation of branched amino acids, biosynthesis of plant hormones like auxin and production of glycine betaine (Corpas et al. 2001). Peroxisomes also maintain a redox balance to counteract the oxidative stress. In peroxisomes, three integral peroxisomal membrane polypeptides (PMPs) having different molecular masses of 18, 29 and 32 kDa were involved in the superoxide production. The NADH acts as the electron donor of 18 and 32 kDa, whereas the 29 kDa acts as the electron donor of NADPH to reduce cytochrome c. Therefore, peroxisomes should be considered as important signalling molecules such as O2 •−, H2O2 and NO˙.
3.6 Heavy Metal Induced Antioxidative Defense System in Plants
Under normal physiological conditions, harmful oxidizing metabolites are produced at an inexpensive level and an appropriate equilibrium exist between the generation and scavenging of ROS that maintain the redox potential of the cell. Exposure of plants to unfavourable environmental conditions such as temperature extremes, heavy metals, drought, water availability, air pollutants, nutrient deficiency, salinity stress perturbed this balance between the production and quenching of ROS, giving rise to rapid increases in intracellular ROS levels (Noctor et al. 2002; Sharma et al. 2010) which can induce oxidative damage to lipids, proteins, and nucleic acids (Fig. 3.5).
The available form of heavy metals for plants uptake are found as soluble components in the soil solution or those solubilised by root exudates (Blaylock and Huang 2000). Plants require certain heavy metals for their growth and upkeep, but excessive amounts of these metals can become toxic to plants and at the same time the ability of plants to accumulate essential metals equally enables them to acquire other nonessential metals (Djingova and Kuleff 2000). As metals are non-biodegradable, they adversely affect the plant both directly and indirectly. When heavy metal concentrations within the plant exceed the optimal level it start showing some direct toxic effects like inhibition of cytoplasmic enzymes and damage to cell structures due to oxidative stress (Jadia and Fulekar 1999). To protect the cellular components from oxidative damage by heavy metal contamination, plants have developed many strategies. Among them antioxidative defense mechanism is the most effective fortification to shield the plant cell (Fig. 3.5). Plants possess very efficient scavenging systems for ROS that protect them from destructive oxidative reactions. These defenses are not restricted to the intracellular compartment, but are also found in the apoplast to a limited extent. Antioxidants are molecules that protect the biomolecules of the cell from being oxidized by free radical reactions thus reduce or obstruct the process of cellular damage. Though protective measures of plants are different from species to species, the presence of antioxidative defense is universal for all that includes both enzymatic and non-enzymatic forms (Tables 3.1 and 3.2).
3.6.1 Enzymatic Antioxidants
3.6.1.1 Superoxide Dismutase
Metalloenzyme superoxide dismutase (SOD) is the most effective intracellular enzymatic antioxidant that plays central role in defense against oxidative damage is found in all aerobic organisms and is susceptible to ROS mediated oxidative damage. The enzyme SOD belongs to the group of metalloenzymes and catalyzes the dismutation of O2 •− to O2 and H2O2.
Three isozymes of SOD (Fig. 3.6) copper/zinc SOD (Cu/Zn-SOD), manganese SOD (Mn-SOD), and iron SOD (Fe-SOD) are reported in plants (Racchi et al. 2001). All forms of SOD are nuclear encoded with an amino terminal targeting sequence that positioned them to their respective subcellular compartments (Bowler et al. 1992). Mn-SOD is localized in mitochondria, whereas Fe- SOD is localized in chloroplasts (Jackson et al. 1978). Cu/Zn-SOD is present in three isoforms, which are found in the cytosol, chloroplast, and peroxisome and mitochondria (del Río et al. 2006). Eukaryotic Cu/Zn-SOD is a dimer and cyanide sensitive whereas the other two (Mn-SOD and Fe-SOD) are cyanide insensitive and may be found as dimer or tetramers forms (del Río et al. 2006). SOD activity has been reported to increase in plants exposed to various environmental stresses, including drought and metal toxicity (Table 3.3). Increased activity of SOD is often associated with increased tolerance of the plant against heavy metal stress. Overproduction of SOD has been reported to result in enhanced oxidative stress tolerance in plants (Gupta et al. 1993). KCN and H2O2 are used for the identification and sensitivity of SOD isozymes while their activity can be detected by negative staining. The Mn-SOD is resistant to both inhibitors; Cu/Zn-SOD is sensitive to both inhibitors whereas; Fe-SOD is resistant to KCN and sensitive to H2O2.
3.6.1.2 Catalase
Under heavy metal stress catalase (CAT), a tetrameric heme containing enzyme is an indispensable detoxifier of ROS as it possess the potential to directly dismutase H2O2 into H2O and O2. It has high affinity for H2O2, but lesser specificity for organic peroxides (R-O-O-R). It has a very high turnover rate (one molecule of CAT can convert ≈6 million molecules of H2O2 to H2O and O2 per minute) and it does not require any reducing equivalent like other antioxidant enzymes. CAT plays an important role in the removal of H2O2 produced by oxidases in peroxisome due to β-oxidation of fatty acids, photorespiration and purine catabolism (Garg and Manchanda 2009). Catalase has been shown to depict variable responses under heavy metal stress (Table 3.4).
3.6.1.3 Ascorbate Peroxidase
Ascorbate Peroxidase (APX) is a fundamental constituent of the Ascorbate-Glutathione (ASC-GSH) cycle, and is thought to play an extremely important role in scavenging ROS and protecting cells in higher plants, algae, euglena and other organisms. APX efficiently scavenges H2O2 in the cytosol and chloroplast exactly in the manner as CAT detoxifies it in peroxisome. The APX reduces H2O2 to H2O and DHA (dehydroascorbate), using Ascorbic acid (AA) as a reducing agent (Fig. 3.7).
Based on locations, viz., cytosolic, mitochondrial, peroxisomal, and chloroplastid (stromal and thylakoidal) and types of amino acid, the APX family is made up of five isomers (Sharma and Dubey 2004). APX has a higher affinity for H2O2 than CAT and POD (peroxidase) and it may have a more crucial role in the management of ROS during stress. Aravind and Prasad (2003) demonstrated increased leaf APX activity under Cd stress in Ceratophyllum demersum. Enhanced activity of APX was also reported by a number of researchers in Brassica juncea, Triticum aestivum and Vigna mungo (Mobin and Khan 2007; Khan et al. 2007; Singh et al. 2008). Hsu and Kao (2004) reported that under non-heat shock conditions pretreatment of Oryza sativa seedlings with H2O2 resulted in an increase in APX activity and protect rice seedlings from subsequent Cd stress. Pekker et al. (2002) studied the expression of APX under iron overload in leaves of de-rooted bean plants and found a rapidly induced expression of APX (mRNA and protein) in response to iron surcharge.
3.6.1.4 Glutathione Reductase
Glutathione reductase (GR) is a flavoprotein oxidoreductase which reduces GSSG (oxidized form of glutathione) to GSH (reduced form of glutathione) by using NADPH as a reductant. Reduced glutathione (GSH) is used up to regenerate AA from Monodehydroascorbate (MDHA) and dehydroascorbate (DHA), and as a result is converted to its oxidized form (GSSG) (Fig. 3.8). GR, a crucial enzyme of ASC-GSH cycle, maintain a high cellular GSH/GSSG ratio by catalyzing the formation of a disulfide bond in glutathione disulfide. It is primarily found in chloroplasts with low amounts occurring in the mitochondria and cytosol. GSH is a low molecular weight compound which reacts with the detrimental ROS members and act as a reductant to prevent thiol groups from being oxidized. GR activity found to be increased in the presence of Cd in Triticum aestivum, Capsicum annuum, Arabidopsis thaliana, Vigna mungo, Brassica juncea (Skorzynska et al. 2004; Khan et al. 2007; Mobin and Khan 2007; Singh et al. 2008).
3.6.1.5 Guaiacol Peroxidase
Guaiacol peroxidase (GPOX) is a heme containing enzyme composed of 40–50 kDa monomers; that eliminates excess H2O2 both during normal metabolism as well as during stress. APX can be distinguished from plant-isolated guaiacol peroxidase (GPOX) in terms of differences in sequences and physiological functions. GPOX degrade indole-3-acetic acid (IAA) and plays a vital role in the biosynthesis of lignin as well as defense against biotic stress by consuming H2O2. GPOX prefers aromatic electron donors such as guaiacol and pyragallol (Asada 1999). The activity of GPOX varies considerably depending upon plant species and conditions of stress. In most of the studies, GPOX activity was found to be enhanced in response to heavy metal stress (Arvind and Prasad 2003; Cho and Seo 2005). While Radotic et al. (2000) noted an initial increase in GPOX activity in spruce needles subjected to Cd stress and subsequent Cd-treatments caused a decline in the activity.
3.6.2 Non-enzymatic Antioxidants
The other half of the antioxidant machinery is comprises of non-enzymatic antioxidants like ascorbic acid, glutathione, α-tocopherol, carotenoids, phenolics, flavonoids, and amino acid cum osmolyte proline. They also play essential role in plant growth and development by influencing cellular processes like mitosis, cell elongation, senescence and cell death.
3.6.2.1 Glutathione
Tripeptide glutathione (glu-cys-gly; GSH) a critical metabolite of plants is considered to be the most important non-enzymatic intracellular defense against ROS induced oxidative damage. It is localized in all cell compartments like cytosol, chloroplast, endoplasmic reticulum, vacuole, peroxisomes, mitochondria, as well as in apoplast and plays a crucial role in numerous physiological processes, including detoxification of xenobiotics, regulation of sulphate transport, expression of stress-responsive genes, signal transduction and conjugation of metabolites. It occurs abundantly in reduced form (GSH) in plant tissues and maintains the normal reducing environment of cells so as to restrain the inhibitory effects of ROS induced oxidative stress (Harinasut et al. 2003).
Enhanced antioxidant activity in the leaves and chloroplast of Phragmites australis was studied by Pietrini et al. (2003). He found its association with a large pool of GSH which resulted in protecting the activity of many photosynthetic enzymes under Cd stress. Increased concentration of GSH has also been observed with the increasing Cd concentration in Pisum sativum by Metwally et al. (2005), Sedum alfredii by Sun et al. (2007) and Vigna mungo by Molina et al. (2008). While Srivastava et al. (2005) reported a significant decline in GR activity and GSH pool under Cu stress.
3.6.2.2 Ascorbic Acid
Ascorbic acid (AA) is the most broadly studied and abundantly found antioxidant compound which is considered to be the most substantial component of a wide range of enzymatic and non-enzymatic reactions as it donates its free electron to them. A minor concentration of AA is being generated by D-galacturonic acid while the remaining considerable amount is produced by Smirnoff-Wheeler pathway in mitochondria, catalyzed by L-galactano-γ-lactone dehydrogenase. AA is the first line of non-enzymatic defense against ROS as 90% of its concentration is found in cytosol and apoplast (Barnes et al. 2002). Demirevska-Kepova et al. (2006) reported that the content of oxidized ascorbate increased during Cd exposure in Hordeum vulgare plants. Contrarily, a decrease in the ASH in the roots and nodules of Glycine max under Cd stress has also been observed (Balestrasse et al. 2001). Cd also decreases the ASH content in Cucumis sativus chloroplast and in the leaves of Arabidopsis thaliana and Pisum sativum (Skorzynska et al. 2004; Romero et al. 2007) whereas, it remained unaffected in Populus Canescens roots (Romero et al. 2007).
3.6.2.3 α -Tocopherol
Vitamin E is an important natural antioxidant of plant leaves, found in the chloroplast envelope, thylakoid membranes and plastoglobuli. Its most common and biologically active form is α-tocopherol. Due to the presence of three methyl groups in its molecular structure, α-tocopherol has the highest antioxidative activity among the four isomers of tocopherols (α, β, γ, δ) found in plants (Kamal and Appelqvist 1996). It is a potent scavenger of photosynthesis-derived reactive oxygen species (mainly 1O2 and OH˙), which check the propagation of lipid peroxidation by deactivating lipid peroxyl radicals in thylakoid membranes. Level of α-tocopherol changes differentially in response to environmental limitations (drought, heavy metals, salinity), intensity of stress and sensitivity to stress. It has been observed that increased production of α-tocopherol results from altered expression, degradation and regeneration of genes that regulate different biochemical pathways, contribute to plant stress tolerance, while decreased levels favor enhanced production of ROS and oxidative damage. Srivastava et al. (2005) reported a general induction in α-tocopherol content in Anabaena doliolum under NaCl and Cu stress. Shuangxia and Daniell (2014), observed that under heavy metal stress, α-tocopherol content in the TMT leaves increased up to 7.5-fold, twice higher than in the wild type.
3.6.2.4 Carotenoids, Flavonoids, Proline and Phenolic Compounds
Carotenoids are the members of lipophilic antioxidant family which are found in the plastids of photosynthetic and non-photosynthetic plant tissues and in microorganisms. Carotenoids unveil their antioxidative activity by protecting the photosynthetic machinery in different ways;
-
(i)
Reacts with lipid peroxidation products to end the chain reactions,
-
(ii)
Prevent the formation of 1O2 as it reacts with 3Chl* and excited chlorophyll (Chl*),
-
(iii)
Scavenge 1O2 and generate heat as a by-product, and
-
(iv)
Dissolve the excess excitation energy, via the xanthophyll cycle.
When excess excitation energy damage the photosynthetic apparatus of plants, flavonoids functions as a secondary ROS scavenging system (Fini et al. 2011). They also have a role in scavenging 1O2 and alleviate the damages caused to the outer envelope of the chloroplast membrane (Agati et al. 2012). Proline, an osmolyte is also regarded as a non-enzymatic antioxidant to prevent the damaging effects of different ROS members. Proline is synthesized using glutamic acid as a substrate, via a pyrroline 5-carboxylate (P5C) intermediate. It is well documented that under heavy metal stress there is a dramatic accumulation of proline in plants. It has been known to act as an osmo-protectant, a metal chelator, a protein stabilizer, an inhibitor of lipid peroxidation, and OH∙ and 1O2 scavenger (Trovato et al. 2008).
Phenolics are secondary metabolites (flavonoids, tannins, hydroxycinnamate esters, and lignin) which possess antioxidant properties. Janas et al. (2009) observed that ROS could serve as a common signal for accumulation to Cu2+ stress and cause accumulation of total phenolic compounds in dark grown lentil roots. Dursun et al. (2016) reported that the application of Cd, Cu, and Pb increased the total phenolics in all treatments of Zea mays.
3.7 Conclusion
It is well documented that almost all plants exhibit a fundamental metal tolerance when exposed to heavy metals. Some species are even capable of hyperaccumulation of heavy metals by employing different tolerance mechanisms as compared to non-accumulating ones. Overall, under normal physiological metabolism, production and scavenging of reactive oxygen species is in dynamic equilibrium state and the involvement of ROS in various metabolic processes might have general implications but when plants are exposed to heavy metal stress they experience overproduction of ROS which adversely affect the plant and ultimately results in oxidative damage. Oxidative stress is a condition in which intra and extracellular compartments of the cells produce considerable amount of ROS or free radicals, which damage the integrity of cells by its toxic effects like membrane lipid peroxidation, protein cross linking, loss of enzyme activity and ion transport, destruction of nucleic acids, lipids and proteins that ultimately leads to its death. However, the cells are equipped with magnificent antioxidative defense system for the fortification of harmful effects of ROS. This chapter gives an insight into how both arms of the antioxidant machinery; either being enzymatic or non-enzymatic, work in synchronicity to diminish the damaging effects of ROS and develop tolerance against heavy metal stress conditions. Although remarkable progress has been achieved in recent years, there are still equivocation and gaps in our understanding of how heavy metals induced the antioxidative defense system of plants. Explanation of the mechanism of plant resistance to heavy metals, and further exploration of key factors controlling heavy metal damage and resistance are the focus of future research, in order to provide important basis for the plant and heavy metal relationship.
References
Adriano DC (2003) Trace elements in terrestrial environments: biogeochemistry, bioavailability and risks of metals, 2nd edn. Springer, New York, pp 1–6
Agati G, Azzarello E, Pollastri S, Tattini M (2012) Flavonoids as antioxidants in plants location and functional significance. Plant Sci 196:67–76
Allen RD, Webb RP, Schake SA (1997) Use of transgenic plants to study antioxidant defences. Free Radic Biol Med 23:473–479
Alloway BJ, Ayres DC (1997) Chemical principles of environmental pollution, 2nd edn. CRC Press, London, p 395
Aravind P, Prasad MNV (2003) Zinc alleviates cadmium-induced oxidative stress in Ceratophyllum demersum L: a free-floating freshwater macrophyte. Plant Physiol Biochem 41:391–397
Arisi ACM, Mocquot B, Mench M, Foyer CH, Jouanian L (2000) Responses to cadmium in leaves of transformed poplars overexpressing γ-glutamylcysteine synthetase. Physiol Plant 109:143–149
Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50:601–639
Asada K (2006) Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol 141:391–396
Axtell NR, Sternberg PKS, Claussen K (2003) Lead and nickel removal using Microspora and Lemna minor. Bioresour Technol 89:41–48
Badiye A, Kapoor N, Khajuria H (2013) Copper toxicity: a comprehensive study. Res J Recent Sci 2:58–67
Baker AJM, Brooks RR (1989) Terrestrial higher plants which hyperaccumulate metallic elements- a review of their distribution, ecology and phytochemistry. Biorecovery 1:81–126
Baker AJM, McGrath SP, Reeves RD, Smith JAC (2000) Metal hyperaccumulator plants: a review of the ecology and physiology of a biological resource for phytoremediation of metal-polluted soils. In: Terry N, Bañuelos G (eds) Phytoremediation of contaminated soil and water. CRC Press, Boca Raton, pp 85–108
Balestrasse KB, Gardey L, Gallego SM, Tomaro ML (2001) Response of antioxidant defence system in soybean nodules and roots subjected to cadmium stress. Aust J Plant Physiol 28:497–504
Barcelo J, Poschenrieder C, Andreu I, Gunse B (1986) Cadmium induced decrease of water stress resistance in bush bean plants (Phaseolus vulgaris L. cv. Contender) I. Effect of Cd on water potential, relative water content, and cell wall elasticity. J Plant Physiol 125:17–25
Barnes J, Zheng Y, Lyons T (2002) Plant resistance to zone: the role of ascorbate. In: Omasa KH, Saji S, Youssefian N (eds) Air pollution and plant biotechnology – prospects for phytomonitoring and phytoremediation. Springer, Tokyo, pp 235–252
Baryla A, Carrier P, Franck F, Coulomb C, Sahut C, Havaux M (2001) Leaf chlorosis in oilseed rape plants (Brasica napus) grown on cadmium polluted soil: cause and consequences for photosynthesis and growth. Planta 212:696–709
Baszynski T, Wajda L, Krol M, Wolinska D, Krupa A, Tuken–Dorf (1980) Photosynthetic activities of cadmium–treated tomato plants. Physiol Plant 48:365–370
Bhattacharjee S (2005) Reactive oxygen species and oxidative burst: roles in stress, senescence and signal transduction in plant. Curr Sci 89:1113–1121
Blaylock MJ, Huang JW (2000) Phytoextraction of metals. In: Raskin I, Ensley BD (eds) Phytoremediation of toxic metals: using plants to clean up the environment. Wiley, New York, pp 53–70
Blaylock MJ, Salt DE, Dushenkov S, Zakharova O, Gussman C, Kapulnik Y, Ensley BD, Raskin I (1997) Enhanced accumulation of Pb in Indian mustard by soil-applied chelating agents. Environ Sci Technol 31(3):860–865
Blokhina O, Fagerstedt KV (2010) Reactive oxygen species and nitric oxide in plant mitochondria: origin and redundant regulatory systems. Physiol Plant 138:447–462
Bowler C, Van Montagu M, Inze D (1992) Superoxide dismutase and stress tolerance. Annu Rev Plant Physiol Plant Mol Biol 43:83–116
Cai-lin GE, Yang X, Yang J (2003) Effect of heavy metal stress on different rice varieties of superoxide dismutase. Acta Agric Nucleatae Sin 17(4):286–291
Chaney RL, Angle JS, Li YM, Baker AJM (1999) Method for phytomining of nickel, cobalt and other metals from soil. US Patent 5,944,872, 27 Jan
Chang TC, You SJ, Yu BS, Chen CM, Chiu YC (2009) Treating high-mercury-containing lamps using fullscale thermal desorption technology. J Hazard Mater 162:967–972
Che D, Meagher R, Heaton ACP, Lima A, Rugh CL, Merkle SA (2003) Expression of mercuric ion reductase in Eastern cottonwood (Populus deltoides) confers mercuric ion reduction and resistance. Plant Biotechnol J 1:311–319
Chen HM, Zeng CR, Tu C, Shen ZG (2000) Chemical methods and phytoremediation of soil contaminated with heavy metals. Chemosphere 41:229–234
Cho U, Seo N (2005) Oxidative stress in Arabidopsis thaliana exposed to cadmium is due to hydrogen peroxide accumulation. Plant Sci 168:113–120
Cleland RE, Grace SC (1999) Voltammetric detection of superoxide production by photosystem II. FEBS Lett 457:348–352
Corpas FJ, Barroso JB, del Río LA (2001) Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci 6:145–150
Cosio C, Martinoia E, Keller C (2004) Hyperaccumulation of cadmium and zinc in Thlaspi caerulescens and Arabidopsis halleri at the leaf cellular level. Plant Physiol 134:716–725
D’Souza R, Varun M, Pratas J, Paul MS (2013) Spatial distribution of heavy metals in soil and flora associated with the glass industry in North Central India: implications for phytoremediation. Soil Sediment Contam 22:1–20
Davis LC, Castro-Diaz S, Zhang Q, Erickson LE (2002) Benefits of vegetation for soils with organic contaminants. Crit Rev Plant Sci 21:457–491
del Río LA, Sandalio LM, Corpas FJ, Palma JM, Barroso JB (2006) Reactive oxygen species and reactive nitrogen species in peroxisomes. Production, scavenging, and role in cell signaling. Plant Physiol 141:330–335
Demirevska-Kepova K, Simova-Stoilova L, Stoyanova ZP, Feller U (2006) Cadmium stress in barley: growth, leaf pigment, and protein composition and detoxification of reactive oxygen species. J Plant Nutr 29:451–468
Djingova R, Kuleff I (2000) Instrumental techniques for trace analysis. In: Vernet JP (ed) Trace elements: their distribution and effects in the environment. Elsevier, London
Dursun K, Elmastaş M, Ozturk L, Kayır O (2016) Responses of the phenolic compounds of Zea mays under heavy metal stress. Appl Biol Chem 59:813–820
Ebinghaus R, Tripathi RM, Wallschläger LSE (1999) Natural and anthropogenic mercury sources and their impact on the air-surface exchange of mercury on regional and global scales. In: Ebinghaus R, Turner RR, Lacerda LD, Vasiliev O, Salomons W (eds) Mercury contaminated sites: characterization, risk assessment and remediation. Springer, New York
Eick MJ, Peak JD, Brady PV, Pasak JD (1999) Kinetics of lead absorption and desorption on goethite: residence time effect. Soil Sci 164:28–39
Elinder CG (1985) Cadmium: uses, occurrence, and intake. In: Friberg L, Elinder CG, Kjellstrom T (eds) Cadmium and health: a toxicological and epidemiological appraisal. CRC Press, Boca Raton, pp 24–63
Elstner EF (1991) Mechanism of oxygen activation in different compartments. In: Pell EJ, Steffen KL (eds) Active oxygen/oxidative stress and plant metabolism. American Socienty of Plant Physiologists, Roseville, pp 13–25
EPA (2004) National priorities list for Smalley-Piper. US Environmental Protection Agency, Collierville http://www.epa.gov/superfund/sites/npl/nar1727.htm
Falandysz J, Lipka K, Kawano M, Brzostowski A, Dadej M, Jedrusiak A, Puzyn T (2003) Mercury content and its bioconcentration factors in wild mushrooms at Lukta and Morag, northeastern Poland. J Agric Food Chem 51:2832–2836
Fini A, Brunettii C, Di Ferdinando M, Ferrini F, Tattini M (2011) Stress induced Flavonoid biosynthesis and the antioxidant machinery of plants. Plant Signal Behav 6:709–771
Fontes RLF, Cox FR (1995) Effects of sulfur supply on soybean plants exposed to zinc toxicity. J Plant Nutr 18:1893–1906
Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866–1875
Foyer CH, Lopez-Delgado H, Dat JF, Scott IM (1997) Hydrogen peroxide- and glutathione-associated mechanisms of acclimatory stress tolerance and signalling. Physiol Plant 100:241–254
Francesconi K, Visoottiviseth P, Sridokchan W, Goessler W (2002) Arsenic species in an arsenic hyperaccumulating fern, Pityrogramma calomelanos: a potential phytoremediator of arsenic contaminated soils. Sci Total Environ 284:27–35
Gardea-Torresdey JL, Peralta-Videa JR, Rosa GD, Parsons JG (2005) Phytoremediation of heavy metals and study of the metal coordination by X-ray absorption spectroscopy. Environ Health Perspect 249:1797–1810
Garg N, Manchanda G (2009) ROS generation in plants: boon or bane? Plant Biosyst 143:8–96
Gratao PL, Polle A, Lea PJ, Azevedo RA (2005) Making the life of heavy metal-stressed plants a little easier. Funct Plant Biol 32:481–494
Gumaelius L, Lahner B, Salt DE, Banks JA (2004) Arsenic hyperaccumulation in gametophytes of Pteris vittata. A new model system for analysis of arsenic hyperaccumulation. Plant Physiol 36:3198–3208
Guo T, Zhang G, Zhou M, Wu F, Chen J (2004) Effects of aluminum and cadmium toxicity on growth and antioxidant enzyme activities of two barley genotypes with different Al resistance. Plant Soil 258:241–248
Gupta AS, Heinen JL, Holaday AS, Burke JJ, Allen RD (1993) Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase. Proc Natl Acad Sci U S A 90:1629–1633
Haag-Kerwer A, Schafer HJ, Heiss S, Walter C, Rausch T (1999) Cadmium exposure in Brassica juncea causes a decline in transpiration rate and leaf expansion without effect on photosynthesis. J Exp Bot 50:1827–1835
Halliwell B (2006) Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life. Plant Physiol 141:312–322
Halliwell B, Gutteridge JMC (1999) Free radicals in biology and medicine. Clarendon Press, Oxford
Hambidge KM, Krebs NF (2007) Zinc deficiency: a special challenge. J Nutr 137:1101–1107
Harinasut P, Poonsopa D, Roengmongkol K, Charoensataporn R (2003) Salinity effects on antioxidant enzymes in mulberry cultivar. Sci Asia 29:109–113
Hasan SA, Hayat S, Ali B, Ahmad A (2008) 28-homobrassinolide protects chickpea (Cicer arietinum) from cadmium toxicity by stimulating antioxidants. Environ Pollut 151:60–66
Heyno E, Mary V, Schopfer P, Krieger-Liszkay A (2011) Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes. Planta 234:35–45
Hsu YT, Kao CH (2004) Cadmium toxicity is reduced by nitric oxide in rice leaves. J Plant Growth Regul 42:227–238
Islam E, Yang X, Li T, Liu D, Jin X, Meng F (2007) Effect of Pb toxicity on root morphology, physiology and ultrastructure in the two ecotypes of Elsholtzia argyi. J Hazard Mater 147:806–816
Jackson C, Dench J, Moore AL, Halliwell B, Foyer CH, Hall DO (1978) Subcellular localisation and identification of superoxide dismutase in the leaves of higher plants. Eur J Biochem 91:339–344
Jadia CD, Fulekar MH (1999) Phytoremediation of heavy metals: recent techniques. Afr J Biotechnol l8:921–928
Janas KM, Amarowicz R, Zielinska-Tomaszewska J, Kosinśka A, Posmyk MM (2009) Induction of phenolic compounds in two dark-grown lentil cultivars with different tolerance to copper ions. Acta Physiol Plant 31:587–595
Kabata-Pendias A (2001) Trace elements in soils and plants. CRC Press, Boca Raton
Kacabova P, Natr L (1986) Effect of Pb on growth characteristics and chlorophyll content in barley seedlings. Phosynthetica 20:411–417
Kahle H (1993) Response of roots of trees to heavy metals. Environ Exp Bot 33:99–119
Kamal-Eldin A, Appelqvist LA (1996) The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids 31:671–701
Karuppanapandian T, Moon JC, Kim C, Manoharan K, Kim W (2011) Reactive oxygen species in plants: their generation, signal transduction, and scavenging mechanisms. Aust J Crop Sci 5:709–725
Kerkeb L, Kramer U (2003) The role of free histidine in xylem loading of nickel in Alyssum lesbiacum and Brassica juncea. Plant Physiol 131:716–724
Khan NA, Samiullah, Singh S, Naza R (2007) Activities of antioxidative enzymes, sulphur assimilation, photosynthetic activity and growth of wheat (Triticum aestivum) cultivars differing in yield potential under cadmium stress. J Agron Crop Sci 193:435–444
Khurana N, Chatterjee C (2001) Influence of variable zinc on yield, seed oil content, and physiology of sunflower. Soil Sci Plant Ann 32:3023–3030
Krieger-Liszkay (2005) Singlet oxygen production in photosynthesis. J Exp Bot 56:337–346
Leon AM, Palma JM, Corpas FJ, Gomez M, Romero-Puertas MC, Chatterjee D, Mateos RM, del Rio LA, Sandalio LM (2002) Antioxidant enzymes in cultivars of pepper plants with different sensitivity to cadmium. Plant Physiol Biochem 40:813–820
Li Y, Song Y, Shi G, Wang J, Hou X (2009) Response of antioxidant activity to excess copper in two cultivars of Brassica campestris ssp. chinensis Makino. Acta Physiol Plant 31:55–162
Liao S, Chang N (2004) Heavy metal phytoremediation by water hyacinth at constructed wetlands in Taiwan. J Aquat Plant Manag 42:60–68
Lösch R (2004) Plant mitochondrial respiration under the influence of heavy metals. In: Prasad MNV (ed) Heavy metal stress in plants from biomolecules to ecosystems, 2nd edn. Springer, Berlin, pp 182–200
Marschner H (1995) Mineral nutrition of higher plants. Academic, London, pp 24–28
Massa N, Andreucci F, Poli M, Aceto M, Barbato R, Berta G (2010) Screening for heavy metal accumulators amongst autochthonous plants in a polluted site in Italy. Ecotoxicol Environ Saf 73:1988–1997
McArthur J, Ravenscroft P, Safiulla S, Thirlwall MF (2001) Arsenic in groundwater: testing pollution mechanisms for sedimentary aquifers in Bangladesh. Water Resour Res 37:109–117
McIntyre T (2003) Phytoremediation of heavy metals from soils. Adv Biochem Eng Biotechnol 78:97–123
Metwally A, Safronova VI, Belimov AA, Dietz KJ (2005) Genotypic variation of the response to cadmium toxicity in Pisum sativum L. J Exp Bot 56:167–178
Minnich MM, McBride MB, Chaney RL (1987) Copper activity in soil solution. II. Relation to copper accumulation in young snap beans. Soil Sci Soc Am J 51:573–578
Mitchell RL, Burchett MD, Pulkownik A, McCluskey L (1988) Effects of environmentally hazardous chemicals on the emergence and early growth of selected Australian native plants. Plant Soil 112:195–199
Mobin M, Khan NA (2007) Photosynthetic activity, pigment composition and antioxidative response of two mustard (Brassica juncea) cultivars differing in photosynthetic capacity subjected to cadmium stress. J Plant Physiol 164:601–610
Molina AS, Nievas C, Chaca MVP, Garibotto F, González U, Marsá SM, Luna C, Giménez MS, Zirulnik F (2008) Cadmium-induced oxidative damage and antioxidative defense mechanisms in Vigna mungo L. Plant Growth Regul 56:285–295
Navrot N, Rouhier N, Gelhaye E, Jaquot JP (2007) Reactive oxygen species generation and antioxidant systems in plant mitochondria. Physiol Plant 129:185–195
Noctor G, Veljovic-Jovanovic S, Driscoll S, Novitskaya L, Foyer CH (2002) Drought and oxidative load in the leaves of C3 plants: a predominant role for photorespiration? Ann Bot 89:841–850
Noctor G, Paepe RD, Foyer CH (2006) Mitochondrial redox biology and homeostasis in plants. Trends Plant Sci 12:125–134
Nouet C, Motte P, Hanikenne M (2011) Chloroplastic and mitochondrial metal homeostasis. Trends Plant Sci 16(7):395–404
O’Sullivan JN, Asher CJ, Blamey FPC (1997) Nutrient disorders of sweet potato, ACIAR monograph no. 48. Australian Centre for International Agricultural Research, Canberra
Ottabbong E (1989) Chemistry of Cr in some Swedish soil: 2. Fate and impact of added Cr on pH and status of soluble Mn in four soils. Acta Agric Scand 39:131–138
Paivoke H (1983) The short term effect of Zn on the growth anatomy and acid phosphate activity of pea seedlings. Ann Bot 20:307–309
Palma JM, Corpas FJ, del Rio LA (2009) Proteome of plant peroxisomes: new perspectives on the role of these organelles in cell biology. Proteomics 9:2301–2312
Patterson W, Olson JJ (1983) Effects of heavy metals on radicle growth of selected woody species germinated on filter paper, mineral and organic soil substrates. Can J For Res 13:233–238
Peer WA, Mamoudian M, Lahner B, Reeves RD, Murphy AS, Salt DE (2003) Identifying model metal hyperaccumulating plants: germplasm analysis of 20 Brassicaceae accessions from a wide geographic area. New Phytol 159:421–430
Pekker I, Tel-or E, Mittler R (2002) Reactive oxygen intermediates and glutathione regulate the expression of cytosolic ascorbate peroxidase during iron mediated oxidative stress in bean. Plant Mol Biol 49:429–438
Pence NS, Larsen PB, Ebbs SD, Letham DL, Lasat MM, Garvin DF, Eide D, Kochian LV (2000) The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Proc Natl Acad Sci U S A 97:4956–4960
Pfannschmidt T (2003) Chloroplast redox signals: how photosynthesis controls its own genes. Trends Plant Sci 8:33–41
Phillips DA (1981) Chemistry and biochemistry of trace metals in biological systems. In: Lepp NW (ed) Effect of heavy metal pollution on plants, vol 1. Applied Science Publishers, London
Pietrini F, Iannelli MA, Pasqualini S, Massacci A (2003) Interaction of cadmium with glutathione and photosynthesis in developing leaves and chloroplasts of Phragmites australis (Cav.) Trin. ex Steudel. Plant Physiol 133:829–837
Poskuta JW, Parys E, Romanovska E (1987) Effects of lead on the gaseous exchange and photosynthetic carbon metabolism of pea seedlings. Acta Bot Soc Poland 57:149–155
Pulford I, Watson C, McGregor SD (2001) Uptake of chromium by trees: prospects for phytoremediation. Environ Geochem Health 23:307–311
Racchi ML, Bagnoli F, Balla I, Danti S (2001) Differential activity of catalase and superoxide dismutase in seedlings and in vitro micropropagated oak (Quercus robur L.). Plant Cell Rep 20:169–174
Radotic K, Ducic T, Mutavdzic D (2000) Changes in peroxidase activity and isoenzymes in spruce needles after exposure to different concentrations of cadmium. Environ Exp Bot 44:105–113
Rasmusson G, Geisler DA, Møller IM (2008) The multiplicity of dehydrogenases in the electron transport chain of plant mitochondria. Mitochondrion 8:47–60
Ren F, Liu T, Liu H, Hu B (1993) Influence of zinc on the growth, distribution of elements, and metabolism of one-year old American ginseng plants. J Plant Nutr 16:393–405
Romero MC, Corpas FJ, Rodrıguez-Serrano M, Gomez M, del Rio LA, Sandalio LM (2007) Differential expression and regulation of antioxidative enzymes by cadmium in pea plants. J Plant Physiol 164:1346–1357
Sahi SV, Bryant NL, Sharma NC, Singh SR (2002) Characterization of a lead hyperaccumulator shrub, Sesbania drummondii. Environ Sci Technol 36:4676–4680
Salim RM, Isa MM, Subu AL, Sayrafi SA, Sayrafi O (1995) Effect of irrigation with lead and cadmium on the growth and metal uptake of cauliflower, spinach and parsley. J Environ Sci Health Part A 30:831–849
Sas-Nowosielska A, Galimska-Stypa R, Kucharski R, Zielonka U, Małkowski E, Gray L (2008) Remediation aspect of microbial changes of plant rhizosphere in mercury contaminated soil. Environ Monit Assess 137:101–109
Scandalios JG (2005) Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defences. Braz J Med Biol Res 38:995–1014
Senger R, Gautam S, Garg SK, Senger K, Chaudhary R (2009) Lead stress effects on physiobiochemical activities of higher plants. Rev Environ Contam Toxicol 196:1–21
Shanker AK, Cervantes C, Loza-Tavera H, Avudainayagam S (2005) Chromium toxicity in plants. Environ Int 31:739–753
Sharma S, Adholeya A (2011) Detoxification and accumulation of chromium from tannery effluent and spent chrome effluent by Paecilomyceslilacinus fungi. Int Biodeterior Biodegrad 65:309–317
Sharma SS, Dietz KJ (2009) The relationship between metal toxicity and cellular redox imbalance. Trends Plant Sci 14:43–50
Sharma P, Dubey RS (2004) Ascorbate peroxidase from rice seedlings properties of enzyme isoforms, effects of stresses and protective roles of osmolytes. Plant Sci 167:541–550
Sharma P, Jha AB, Dubey RS (2010) Oxidative stress and antioxidative defense system in plants growing under abiotic stresses. In: Pessarakli M (ed) Handbook of plant and crop stress. CRC Press, Boca Raton, pp 89–138
Sharma P, Ha ABJ, Dubey RS, Pessarakli M (2012) Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot 2012:217037. https://doi.org/10.1155/2012/217037
Shuangxia J, Daniell H (2014) Expression of γ-tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal-induced oxidative stress by reducing reactive oxygen species. Plant Biotechnol J 12:1274–1285
Shukla V, Dhankar M, Prakash J, Sastry KV (2007) Bioaccumulation of Zn, Cu and Cd in Channa punctatus. J Environ Biol 28:395–397
Singh S, Khan NA, Nazar R, Anjum NA (2008) Photosynthetic traits and activities of antioxidant enzymes in blackgram (Vigna mungo L. Hepper) under cadmium stress. J Plant Physiol 3:25–32
Skorzynska PE, Drazkiewicz M, Krupa Z (2004) The activity of the antioxidative system in cadmium-treated Arabidopsis thaliana. Biol Plant 47:71–78
Skovsen E, Snyder JW, Lambert JD, Ogilby PR (2005) Lifetime and diffusion of singlet oxygen in a cell. J Phys Chem B 109:8570–8573
Srivastava PC, Gupta UC (1996) Trace elements in crop production. Science Publishers, Lebanon
Srivastava AK, Bhargava P, RaiL C (2005) Salinity and copper-induced oxidative damage and changes in antioxidative defense system of Anabaena doliolum. World J Microb Biotechnol 22:1291–1298
Sujatha P, Gupta A (1996) Tannery effluent characteristics and its effects on agriculture. J Ecotoxicol Environ Monit 6:45–48
Sun Q, Yec ZH, Wang XR, Wong MH (2007) Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii. J Plant Physiol 164:1489–1498
Tan YF, O'Toole N, Taylor NL, Millar AH (2010) Divalent metal ions in plant mitochondria and their role in interactions with proteins and oxidative stress-induced damage to respiratory function. Plant Physiol 152:747–761
Tomiyasu T, Kodamatani H, Imura R, Matsuyama A, Miyamoto J, Akagi H, Kocman D, Kotnik J, Fajon V, Horvat M (2017) The dynamics of mercury near Idrija mercury mine, Slovenia: horizontal and vertical distributions of total, methyl, and ethyl mercury concentrations in soils. Chemosphere 184:244–252
Triantaphylids C, Krischke M, Hoeberichts FA, Ksas B, Gresser G, Havaux M (2008) Singlet oxygen is the major reactive oxygen species involved in photooxidative damage to plants. Plant Physiol 148:960–968
Trovato M, Mattioli R, Costantino P (2008) Multiple roles of proline in plant stress tolerance and development. Rend Lincei 19:325–346
USEPA (2000) A guide to developing and documenting cost estimates during the feasibility study (EPA 540-R-00-002). http://www.epa.gov/superfund/resources/remedy/costest.html
Valavanidis A, Vlachogianni T (2010) Metal pollution in ecosystems: ecotoxicology studies and risk assessment in the marine environment. Sci Adv Environ Toxicol Ecotoxicol Issues 1–14
Varun M, D’Souza R, Pratas J, Paul MS (2012) Metal contamination of soils and plants associated with the glass industry in North Central India: prospects of phytoremediation. Environ Sci Pollut Res 19:269–281
Varun M, Jaggi D, D’Souza R, Paul M, Kumar B (2015) Abutilon indicum L.: a prospective weed for phytoremediation. Environ Monit Assess 187(8):527. https://doi.org/10.1007/s10661-015-4748-3
Vogel-Mikus K, Drobne D, Regvar M (2005) Zn, Cd and Pb accumulation and arbuscular mycorrhizal colonisation of pennycress Thlaspi praecox Wulf. (Brassicaceae) from the vicinity of a lead mine and smelter in Slovenia. Environ Pollut 133:233–242
Wang J, Zhao F, Meharg AA, Raab A, Feldmann J, McGrath SP (2002) Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics, interactions with phosphate, and arsenic speciation. Plant Physiol 130:1552–1561
Wei CY, Chen TB (2006) Arsenic accumulation by two brake ferns growing on an arsenic mine and their potential in phytoremediation. Chemosphere 63:1048–1053
Xie F, Wang H, Wang H (2009) Effects of arsenic on activities of antioxidant enzymes in the fronds of plants with different abilities to accumulate arsenic. J Agro-Environ Sci 28(7):1379–1385
Yan Y, Jian-Ping L, Xue-Hong Z (2008) Response of hyperaccumulator Leersia hexandra Swartz. to chromium-induced oxidative stress. Ecol Environ 17(4):1476–1482
Yan L, Liuke-Hui, Yu F (2011) Effects of manganesee on enzymatic and non-enzymatic antioxidative defenses of the hyperaccumulate Ploygonum pubescens Blume. J Agro-Environ Sci 30:2422–2427
Yang XE, Long XX, Ni WZ, Ye ZQ, He ZL, Stoffella PJ, Calvert D (2002) Assessing copper thresholds for phytotoxicity and potential dietary toxicity in selected vegetable crops. J Environ Sci Health 37:625–635
Yang G, Liang M, Zhou P (2010) Antioxidative defense system differences among four plants under combined Pb and Cd stress. Chin J Eco-Agric 18(4):836–842
Zavoda J, Cutright T, Szpak J, Fallon E (2001) Uptake, selectivity, and inhibition of hydroponic treatment of contaminants. J Environ Eng 127:502–508
Zhao F, Lombi E, Breedon T, McGrath SP (2000) Zinc hyperaccumulation and cellular distribution in Arabidopsis halleri. Plant Cell Environ 23:507–514
Zhu B, Alva AK (1993) Effect of pH on growth and uptake of copper by Swingle citrumelo seedlings. J Plant Nutr 16:1837–1845
Zhu Q, Xia H (2012) Effects of lead stress on antioxidant enzyme system and chlorophyll content of Pteris vittata. Guizhou Agric Sci 40:56–58
Zhu YL, Zayed AM, Quian JH, D’souza M, Terry N (1999) Phytoaccumulation of trace elements by wetland plants: II. Water hyacinth. J Environ Qual 28:339–344
Acknowledgements
Financial support from University Grants Commission [UGC-MRP – F. No. 43-100/2014(SR)] is gratefully acknowledged. We gratefully acknowledge the University Grants Commission for providing financial support by sanctioning the Post Doctoral Fellowship (F./PDFSS-2014-15-SC-UTT-8854) to Mayank Varun.
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Ishtiyaq, S., Kumar, H., Varun, M., Kumar, B., Paul, M.S. (2018). Heavy Metal Toxicity and Antioxidative Response in Plants: An Overview. In: Hasanuzzaman, M., Nahar, K., Fujita, M. (eds) Plants Under Metal and Metalloid Stress. Springer, Singapore. https://doi.org/10.1007/978-981-13-2242-6_3
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