Keywords

6.1 Introduction

Halophytic plants are a group of plants that can grow and survive under high salinity conditions. These plants need saline habitat to complete their life and grow naturally on saline soil (Rozema et al. 2013). According to Flowers and Colmer (2008), halophytes normally survive at 200 mM NaCl and can tolerate even higher salt concentrations. Halophytic plants can be grown on soil and water that are unsuitable for glycophytes (Flowers et al. 2010). Plants are exposed to different kinds of stresses like drought, salinity, high temperature, heavy metals, toxic ions, etc., and the major reason behind these toxicities may be human activities or natural abundance. Heavy metals are of more concern because they are nondegradable and have a longer half life, i.e. persist for longer time in the environment. The trace elements which have a density of more than 4 g/cm3 are defined as heavy metals (Hawkes 1997). Trace elements of periodic table include both essential and non-essential elements along with toxic heavy metals. For example, copper is an essential micronutrient involved in various physiological reactions via Cu2+ enzymes, while cadmium is a non-essential heavy metal causing the disturbances in cell functioning. Cd2+ ions interact with thiol groups of cell constituents and result in growth reduction by disturbing photosynthesis, ion and water transport as well as enzymatic activities. Heavy metals not only affect the plant growth and productivity but also the human health (Lopez-Climent et al. 2011). High levels of heavy metals result in the oxidative stress through the production of reactive oxygen species (Mittler 2002). ROS react with other cellular components like lipids, proteins, photosynthetic pigments and DNA which results in membrane damage, lipid peroxidation, degradation of metabolites, inactivation of enzymes and even cell death (Nagarani et al. 2012). Heavy metals may interact with proteins through affinity for S and N atoms in side chains of amino acids (Wei et al. 2003). They can also bind with the sulphydryl groups of enzymes and structural proteins. Heavy metals cations can displace an essential cation in specific binding sites (Sharma and Dietz 2009).

Halophytes are many times exposed to heavy metal toxicity particularly in the mangrove ecosystem. They act as a major sink for a number of pollutants from nearby drainage and rivers of mangrove-growing area (MacFarlane and Burchett 2001). Heavy metal pollution of mangrove ecosystem in India is due to urban and agricultural runoff, industrial waste, boating and recreational activities including chemical spills, leachates from domestic garbage dumps, sewage water treatment plants and mining, etc. (Lokhande et al. 2011). Many estuary salt marshes and large area of oasis farmlands of arid and semiarid regions are affected by both salinity and heavy metal stress (Defew et al. 2005; Han et al. 2012). In India, ship scraping industry also causes pollution by producing waste products like crude petroleum and heavy metals as byproducts (Tewari et al. 2001; Srinivasa Reddy et al. 2003). Banerjee et al. (2017) reported that Chilika Lake of Orrisa (India) is polluted by heavy metals like Hg, Cd, Co and Pb. Shackira and Puthur (2013) detected significantly higher concentrations of Cd than other heavy metals in the sediment and the water samples in Kadalundi-Vallikkunnu Community Reserve wetlands. Mandal et al. (2009) have reported arsenic pollution in West Bengal (Sunderban region) due to industrial effluents, agricultural runoff and sewage discharge.

The halophytic plants possess morphological, anatomical and physiological adaptations allowing them to survive in soils with high concentrations of salinity (Flowers and Colmer 2008). These adaptations include ion compartmentalization, osmotic adjustment, ion transport and uptake, redox energy balance, activation of antioxidative defence system, succulence, salt accumulation or extrusion (Lokhande and Suprasanna 2012). These adaptations help halophytic plants to cope with other abiotic stresses like temperature, high light intensities, heavy metals, etc. (Lopez-Chuken and Young 2005; Ruan et al. 2010; Lokhande and Suprasanna 2012; Walker et al. 2014; Panda et al. 2017). There are some specific adaptations to maintain oxidative status in halophytic plants as compared to glycophytic plants to tolerate saline environments (Ellouzi et al. 2011). A better antioxidant system enables them to tolerate heavy metal stress also. Therefore, it has been proposed that heavy metal-accumulating plants and halophytic plants share a number of common processes (Shevyakova et al. 2003). Oxidative metabolism-induced tolerance mechanism has been studied under heavy metal stress in halophyte Salicornia brachiata (Sharma et al. 2010), Kosteletzkya virginica (Han et al. 2012, 2013), Atriplex halimus L. (Bankaji et al. 2016) and S. maritima (Panda et al. 2017). Halophytes have been proposed as potential candidates for removal of heavy metals from contaminated soil due to common tolerance and detoxification mechanisms (Manousaki and Kalogerakis 2009). Ruan et al. (2008) reported that perennial halophyte Kosteletzkya virginica could be utilized for revegetation of salt-affected coastal tidal flats. These studies further confirm that the salt-tolerant species can better adapt to adverse environmental conditions including heavy metal stress (Chai et al. 2013; Lokhande et al. 2011). In general, plants have different mechanism to control heavy metal uptake. These include chelation and sequestration of heavy metal ions by specific ligands to maintain them at low and lesser harmful levels in the cell cytosol (Sbartai et al. 2012). The main chelating substances are phytochelatins (PCs) and metallothioneins, and this ubiquitous detoxification strategy has been described in a number of plants (Yadav 2010).

In the present chapter we will discuss the heavy metal-induced oxidative stress and their interaction with salinity in halophytes and how the antioxidant defence system helps in detoxification of heavy metals. Sulphur metabolism and osmotic adjustment in halophytes in response to heavy metals will also be discussed in detail.

6.2 Oxidative Metabolism in Halophytes Under Heavy Metal Stress

The halophytic plant displays a cascade of events upon heavy metal stress exposure that leads to disturbances in metabolic behaviour. These events include water deficit-induced stomatal closure, limited CO2 availability, over-reduction of ETC chain in chloroplast (photosynthesis) and mitochondria (respiration) and generation of ROS (Lokhande and Suprasana 2012) causing oxidative stress. Heavy metal stress causes the imbalance of the redox metabolism and leads to oxidative stress (Schutzendubel and Polle 2002; Mittler et al. 2004). Oxidative metabolism will be studied under following subheadings.

6.2.1 Reactive Oxygen Species (ROS) Production

The overproduction of ROS like superoxide anion (O2 −), hydrogen peroxide (H2O2) and hydroxyl radical (OH−) resulting in oxidative stress is among the initial heavy metal toxicity responses (Shahid et al. 2014). ROS are highly toxic, and their high concentration can oxidize cell constituents like DNA, carbohydrates, proteins and lipids (Mittler 2002; Mittler et al. 2010). The first step during the O2 reduction is production of relatively short-lived, not readily diffusible hydroperoxyl (HO2 −) and superoxide anions (O2 −). Superoxide anions are very active and can oxidize specific amino acids like histidine, methionine and tryptophan (Lutts and Lefevre 2015). Further it may undergo spontaneous or enzymatic dismutation to produce H2O2; otherwise it may interact with plastocyanin or cytochrome f and reduce them, resulting in a superoxide-mediated cyclic electron flow around PS I (Hormann et al. 1993). H2O2 is a natural toxic plant metabolite, produced as a product of photorespiration. Both O2 − and H2O2 cause membrane damage by attacking membrane lipids (Willenkens et al. 1995). The biological toxicity of H2O2 is due to oxidation of SH-group which is enhanced by a metal catalyst. Hydroxyl radical (OH−) is the most toxic ROS, due to high affinity for the biological molecules which results in oxidative damage to proteins and nucleic acids and induces lipid peroxidation (Demidchik 2014).

Among the heavy metals, Cu, Cr, Fe2+, V and Co produce ROS (O2 −, H2O2, OH−) via Fenton-type reactions. Different researchers documented cellular injury by this type of mechanism like for Fe (Halliwell and Gutteridge 1986), copper (Li and Trush 1993), vanadate (Shi and Dalal 1993) and Cr (Shi et al. 1993). The other heavy metals also known as non-redox metals generate oxidative stress by indirect mechanisms, such as NADPH oxidase-dependent H2O2 accumulation and O2 − formation in mitochondria and hydroperoxy fatty acids (Garnier et al. 2006). These non-redox heavy metals can cause indirect production of ROS through depletion of antioxidant pools (Hossain et al. 2012).

Han et al. (2013) reported nearby sixfold increase in O2 − and threefold increase in H2O2 content in Cd (5 μM)-treated K. virginica plants; however salinity along with Cd reduced the toxic effects of Cd. H2O2 content increased with heavy metal treatments of Cd2+, Ni2+ and As3+ in Salicornia brachiata (Sharma et al. 2010). A 1.5-fold increase with Cd (200 and 300 μM), 2.5-fold with Ni (200 μM) and no survival with 200 μM As3+ were reported. Panda et al. (2017) reported that levels of O2 − go parallel with the SOD activity, i.e. with increase of superoxide O2 − concentration, the SOD activity also increases indicating utilization of SOD in dismutation. Increase in ROS under heavy metals has been observed in halophytic plants Kandelia candel (Zhang et al. 2007), Avicennia marina (MacFarlane and Burchett 2001; Caregnato et al. 2008) and Spartina alterniflora (Chai et al. 2013). However Panda et al. (2017) reported that high concentration of salt (600 mM NaCl) and NaCl in combination with arsenic (As) resulted in a decline in O2 − and H2O2 content in halophyte S. maritima seedlings. Vromman et al. (2016) also reported no significant changes of H2O2 level in A. atacamensis subjected to salinity and As treatments.

ROS have been considered as dangerous molecules and their levels need to be kept as low as possible. However many studies have proposed that ROS also play an important role in the plant defence system against pathogens (Bolwell et al. 2002) and PCD (programmed cell death) (Fath et al. 2002; Demidchik et al. 2010; Demidchik 2014) and also act as intermediate signalling molecule to regulate expression of genes (Neill et al. 2002; Vranova et al. 2002). In the view of multiple functions of these ROS, it is necessary to regulate their level in cell but not to eliminate them from cell completely. The role of H2O2 as a signalling molecule under abiotic stress including heavy metals has been reported by many researchers (Dat et al. 2000; Sexena et al. 2016). Salinity stress-induced ROS production in halophytes may be compensated by various adaptations including ion compartmentalization, strong antioxidant defence system, etc. Heavy metal stress also increases ROS production, and saline adaptation further helps them to cope with heavy metal stress. Sharma et al. (2010) reported reduced level of ROS in Salicornia brachiata plants treated with both heavy metal and salinity as compared to the plants treated with heavy metals only. Han et al. (2013) also reported no significant increase in O2 − and H2O2 in K. virginica plants under salinity and salinity along with Cd treatments. ROS content decreased significantly in S. maritima when treated with high salinity in combination with heavy metal (As+NaCl), suggesting the presence of active and efficient ROS scavenging system present in this halophyte. The decreased level of H2O2 under combined treatment of As+NaCl may be attributed to the cumulative effects of both enzymatic and non-enzymatic antioxidants (Panda et al. 2017). On the other hand, Rangani et al. (2016) reported that significant levels of H2O2 in S. maritima under heavy metal (As) and/or salinity stress may be demonstrating its role in stress signalling.

6.2.2 Membrane Damage

High concentration of ROS in the cellular environment damages the cell structure and biomolecules by affecting cell membrane lipids, proteins and DNA resulting in lipid peroxidation (Schickler and Caspi 1999; Baccouch et al. 2001). Extent of lipid peroxidation is measured in terms of MDA content, a decomposition product of polyunsaturated fatty acids. ROS causes elimination of hydrogen from unsaturated fatty acids which lead to the formation of lipid radical. Lipid structure is destroyed due to a cascade of cyclic reactions leading to respective and lipid acid aldehydes and results in membrane deterioration. The lipid peroxidation leads to dimerization and polymerizations of membrane proteins which is considered to be most damaging to cell membranes (Demidchik 2014). This causes irreversible damage to cell membrane proteins and lipids and makes them leaky and alters their functioning (da Silva 2010). Shackira and Puthur (2017) observed significant increase of twofold in MDA content in roots of halophyte Acanthus ilicifolius L. plants, upon treatment with Cd. Chai et al. (2013) reported significant increase in MDA content and membrane electrolyte leakage when exposed to Cu2+ more than 200 mg kg−1 soil in halophyte Spartina alterniflora (Poaceae). Similarly an increase of lipid peroxidation under heavy metal stress condition has been reported by many workers like Tao et al. (2012) in mangrove, Bruguiera gymnorrhiza, Han et al. (2013) in K. virginica and Sai Kachout et al. (2015) in A. rosea. Bankaji et al. (2016) reported 1.6-fold and 5.9-fold increased MDA in root tissue of halophyte Atriplex halimus when irrigated with Cd (400 μM) and Cu (400 μM) solution, respectively, indicating oxidative damage-induced lipid peroxidation. Addition of 200 mM NaCl to the irrigation medium along with Cu2+ and Cd resulted in significantly lower values of MDA, i.e. NaCl treatment showed their protective effect in reducing oxidative damage caused by heavy metals. Similarly Han et al. (2012) also reported that the presence of NaCl reduced to some extent the oxidative stress-induced membrane damage in cadmium-treated plants that was manifested by less lipid peroxidation and protein oxidation. 50 mM NaCl treatment in the nutrient solution for 2 weeks had no influence on MDA content as compared to control plants in halophyte K. virginica, but an increase was observed with Cd treatment of 5 μM (Han et al. 2013). A reduction of about 25% in MDA content was observed when Cd treatment (5 μM) was given along with NaCl (50 mM), showing positive effects of exogenous NaCl on plant responses to Cd2+. This effect has been reported in other halophytes A. halimus and Spartina alterniflora also (Lefevre et al. 2009; Chai et al. 2013). The effect may be due to the dilution caused by salinity-induced growth stimulation in halophytes at moderate concentrations. There are many reports on significant increase in the MDA content in different plants when exposed to high salinity or As stress (Shaheen et al. 2013; Zhao et al. 2010; Tripathi et al. 2012). However Panda et al. (2017) reported no significant change in lipid peroxidation levels (MDA content) in S. maritima plants under salinity, As or combination of both. The total protein content and carbohydrate content in shoot parts of S. salsa seedlings upon zinc exposure were significantly decreased showing the upregulated protein biodegradation (Wu et al. 2013).

6.3 Antioxidant Defence System in Halophytes Under Heavy Metal Stress

Salinity- and heavy metal stress-induced oxidative damage is controlled by antioxidative defence system. This system is composed of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and enzymes of ascorbate-glutathione cycle including ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) and antioxidative metabolites like ascorbate, glutathione, carotenoids, etc. The antioxidative defence system counteracts or neutralizes the harmful effects of ROS (Foyer and Noctor 2005; Asada 2006; Lokhande et al. 2011; Dar et al. 2017). Plants tend to increase phytochelatins synthesis (specific metal-binding proteins), glutathione and polymers of glutathione (precursors for phytochelatins synthesis) as a protective mechanism under heavy metal stress (Tripathi et al. 2007; Begum et al. 2016). Understanding the detoxification mechanism adopted by plants to alleviate or reduce oxidative stress is the key information to further manipulate heavy metal tolerance in halophytic plants. There is difference in spatial localization and biochemical properties of different antioxidative enzymes and metabolites. They also vary in terms of gene expression, and all these properties give a flexible and versatile defence system to cope with excess ROS levels (Vranova et al. 2002). Antioxidant enzymes reduce oxidative stress by interrupting the cascades of uncontrolled oxidation of membrane lipids by oxygen free radicals. Antioxidative enzymes may remove, neutralize or scavenge ROS and their intermediates (Foyer and Noctor 2005). A direct correlation among enzymatic and non-enzymatic antioxidant defence system capacity and salinity stress tolerance has been observed in several halophytic plant species like Crithmum maritimum, Crithmum maritime, Sesuvium portulacastrum, Plantago genus and Mesembryanthemum crystallinum (Ben Amor et al. 2005; Jitesh et al. 2006; Sekmen et al. 2007; Ashraf 2009; Lokhande et al. 2010, 2011). A well-expressed antioxidative defence mechanism in halophytes also helps in overcoming the heavy metal-induced oxidative stress. The various studies on the antioxidant response under heavy metal stress have been listed in Table 6.1. The role of antioxidative enzymes and metabolites are discussed below:

Table 6.1 Examples of halophytic plant species showing the antioxidant defence system in response to heavy metal stress
Full size table

6.3.1 Superoxide Dismutase

Superoxide dismutase (SOD) (EC 1.15.1.1) is a metalloprotein and occurs universally in all plants in aerobic conditions. It acts as primary/first-line defence against ROS-induced damages (Liang et al. 2003). It catalyses the dismutation of O2 − to H2O2 and molecular oxygen (Bowler et al. 1992; Hernandez et al. 2000; Gratao et al. 2005). It acts as a key antioxidative enzyme in aerobic cells as its activity determines the concentration of Haber-Weiss reaction substrate, i.e. superoxide radical and hydrogen peroxide (Sabarinath et al. 2009; Gill et al. 2015). This dismutation reaction is 1000 times faster than the spontaneous dismutation (Gill and Tuteja 2010; Das and Roychoudhury 2014). In plants three classes of SOD have been reported so far based on the metal cofactor. These are di-nuclear Cu/Zn SOD, mononuclear Fe SOD and Mn SOD (Perry et al. 2010). In plants Cu/Zn SOD isoforms are found primarily in the cytosol and chloroplast, Mn SODs are located in the matrix of mitochondria and peroxisomes and Fe SODs are located in the chloroplast (Racchi et al. 2001; Alscher et al. 2002). The upregulation of SOD activity in response to various stresses plays an important and crucial role in alleviation of oxidative stress for the plant survival (Boguszewska et al. 2010). SOD activity increased with increase in production of ROS (Abedi and Pakniyat 2010). SOD activity has been observed to increase in number of halophytes when exposed to different heavy metals.

Sharma et al. (2010) reported in halophytic plant Salicornia brachiata a twofold increase in SOD with Cd2+ (300 μM) and Ni2+ (300 μM) and 1.2-fold with As3+ (100 μM) treatment proving that SOD acts as a first line of defence. In K. virginica plants, Cd (5 μM)-induced SOD activity by twofold as compared to control plants and NaCl alone had a slight promotion in total SOD activity (Han et al. 2013). A twofold increase in SOD activity was observed in plant treated with both Cd and NaCl. This can be correlated with control of oxidative stress produced at cellular level under salt and Cd stress (Hernandez et al. 2001). Similar enhanced activity of SOD under increased concentration of Cd has been reported in halophytic plants like Kandelia candel and Bruguiera gymnorrhiza (Zhang et al. 2007), Spartina densiflora (Redondo-Gomez et al. 2011), Salicornia brachiata (Wang et al. 2014), Atriplex halimus L. (Mesnoua et al. 2016). da Silva (2010) reported slight increase in SOD activity in C3 halophyte limited to coastal salt marshes Halimione portulacoides up to 30 PSU of NaCl and 200 μM Zn treatment. Higher salinities (40 and 50 PSU) result in impaired ROS scavenging system with decreased SOD activity. Transgenic plants overexpressing SOD genes were found tolerant to oxidative stress in maize (Breusegem et al. 1999), salt stress in Arabidopsis (Wang et al. 2004) and drought stress in rice (Yang et al. 2009). Panda et al. (2017) reported a decrease of around 30% SOD activity under As (200 μM and 400 μM), NaCl (200 and 600 mM) and a combination of both (200 μM As+200 mM) treatment in S. maritima as compared to control plants. However, decrease in SOD activity in this halophyte under stress condition indicates its utilization in sequestration of stress-induced O2 − radicals (Rangani et al. 2016). Wu et al. (2013) reported a slight increase in SOD activity with Pb, Zn and their combination in Suaeda salsa. The SOD dismutase O2 − into H2O2 and H2O2 is further scavenged by other antioxidative enzymes like CAT and APX to reduce toxic effect of H2O2 on plants (Bose et al. 2014).

6.3.2 Catalase

Catalase (CAT) (EC 1.11.1.6) is a heme-containing enzyme that catalyses the conversion of H2O2 into H2O and O2 (Das and Roychoudhury 2014). Hydrogen peroxide (H2O2) concentration is expected to increase under normal and abiotic stress conditions either due to action of SOD or by biochemical pathways that directly produce it. H2O2 also acts as signalling molecule and takes part in a number of important functions of plant cell (Foyer and Noctor 2005). But there should be control on its buildup and total concentration in plant cell to protect from oxidative damage to membranes and proteins. Peroxisomes are the major sites of H2O2 production due to β-oxidation of fatty acids, photorespiratory oxidation and purine catabolism (Mittler 2002). There are different forms of catalase isozymes found in different cellular organelles, such as CAT 1 found in peroxisomes and cytosol which is expressed in pollens and seeds, CAT 2 expressed mainly in leaves but also in roots and seeds and CAT 3 localized in mitochondria and which is highly expressed in seeds and seedlings (Scandalias 1990; Sharma et al. 2012). Under the stressful environmental conditions, energy consumption along with increased production of H2O2 increases catabolism. CAT eliminates the H2O2 in an energy-efficient manner without consuming cellular reducing equivalents. According to Sandalio and Romero-Puertas (2015), catalase activity is mostly associated with peroxisome where it is involved in removal of H2O2 mainly formed during photorespiration.

Wu et al. (2013) reported an increase in CAT activity with an upregulation of CAT gene expression in response to Zn (100 μg L−1), Pb (20 μg L−1) and their combined effect in shoots of Suaeda salsa. Panda et al. (2017) observed a significant increase in CAT activity under As (200 μM and 400 μM), NaCl (200 and 600 mM) and a combination of both (200 μM As+200 mM NaCl) treatments with respect to control in S. maritima. A 142% increase was reported in seedlings treated with 400 μM arsenic. There is a negative correlation between activity of CAT and H2O2 concentration in cell showing an effective sequestration of H2O2 under stress conditions. Similarly increased CAT activity has also been observed in other halophytes when exposed to salinity and heavy metal stress Kandelia candel and Bruguiera (Zhang et al. 2007) and salinity Salvadora persica (Rangani et al. 2016). Salt stress (NaCl) alone had no effects on enzyme activity in K. virginica plants; in contrast Cd stress resulted in a threefold reduction in CAT activity. A sixfold decrease as compared to control plant was observed in CAT activity of plants treated with both Cd and NaCl (Han et al. 2013). The Cd-induced decrease in CAT activity has also been observed in halophyte Salicornia brachiata (Parida and Jha 2010), Suaeda salsa (L.), Kochia scoparia (L.) and Beta vulgaris (L.) (Takagi and Yamada 2013). Bankaji et al. (2016) also reported a significant decrease in CAT activity of Atriplex halimus (86.3–64.2%) when treated with Cd2+ or Cu2+ (400 μM), respectively. They reported a decrease in activity when Cd and NaCl (200 mM) were applied simultaneously to the growing medium as compared to those when applied individually. Similar responses were observed with Cu2+ in place of Cd2+ as a heavy metal. Similar increased activity of CAT antioxidant enzymes with increased concentration of Cd has been reported in other halophytic plants including Atriplex halimus L. (Mesnoua et al. 2016) and Salicornia brachiata (Sharma et al. 2010; Wang et al. 2014). The catalase activity increased with 20 PSU and 400 μM Zn treatment (da Silva 2010) in coastal salt marshes Halimione portulacoides. Sharma et al. (2010) reported 1.5- and fivefold increases in CAT activity with Cd (50 μM) and Ni and Zn (200 μM) representing its role in removing heavy metal-induced H2O2 levels.

6.3.3 Peroxidase

Peroxidase (POX) (EC 1.11.1.7) is a heme chloroplastic enzyme that detoxifies H2O2 in the cytosolic compartment of the cell under normal as well as stress conditions (Das and Roychoudhury 2014). They are non-specific in utilizing electrons donors for oxidation of H2O2. They use aromatic compounds like guaiacol and pyrogallol (Asada 1999) as reducing agents. MacFarlane and Burchett (2001) reported a significant increase of 21% in POX activity when exposed to Cu (200 μM) and increased proportionally up to 800 μM Cu treatment in a mangrove Avicennia marina indicating role of peroxidase in ROS amelioration. POX activity also increased under Pb and Zn treatments in Avicennia marina. Peroxidase plays an important and active role in the cytosol, vacuoles and also cell walls and is considered as an essential enzyme for the removal of H2O2 from cell (Das and Roychoudhury 2014). Wu et al. (2013) observed increased POX activity only with Zn treatment (100 μg/L) and not in Pb (20 μg L−1). Similarly increase in POX activity was reported with Cd and Pb combined treatment not with individual treatments (Manonusaki and Kalogerakis 2009). Salt alone has no effect on enzyme activity in K. virginica plants (Han et al. 2013). However an increase of about 87% was observed in POX activity in Cd-treated plants. In the plants treated with both Cd and NaCl, POX activity was maintained at control levels. In S. maritima no significant change in POX activity was observed in NaCl-treated seedling, while As treatment enhanced the activity. A combined treatment of 200 μM As and 200 mM NaCl resulted in 54% increase in POX activity with respect to control (Panda et al. 2017) indicating the role of POX in antioxidative defence mechanism.

6.3.4 Ascorbate Peroxidase

Ascorbate peroxidase (APX) (EC 1.11.1.11) is a key enzyme of the ascorbate-glutathione cycle and catalyses the conversion of H2O2 to water. The ascorbate-glutathione cycle includes two antioxidant metabolites ascorbate and glutathione and enzymes APX, GR, DHAR and MDHAR. The activation of ascorbate-glutathione cycle is essential to control oxidative stress in plants (Gill and Tuteja 2010). It is an efficient pathway which detoxifies H2O2 by performing APX-induced dismutation of H2O2 into H2O and DHA by using ascorbic acid as a reducing agent (Das and Roychodhury 2014). This mechanism was also found important under heavy metal stress (Tiryakioglu et al. 2006; Liu et al. 2007). Ascorbate peroxidase is the primary H2O2 scavenging enzyme in the chloroplast and cytosol of the plant cells (Asada 1999). APX uses ascorbate as it reduces substrate, and there are several isozymes of APX located in different cellular organelles like cytosol, microbodies, chloroplast, etc. (Madhusudhan et al. 2003; Pandey et al. 2017), and they are known with the name of their respective cellular locations like cytosolic, mitochondrial APX, etc. (Noctor and Foyer 1998; Sharma and Dubey 2004). APX acts by a series of coupled reactions (Asada 1994). It plays an important role in ROS scavenging as compared to other antioxidative enzymes as ascorbate, in addition to reacting with H2O2, may react with superoxide anion, singlet oxygen and hydroxyl radical (Shigeoka et al. 2002). Heavy metal-induced APX activity has been reported widely in literature (Sheokand et al. 2010; Kumari et al. 2017) under salinity (Kumari et al. 2010) and heavy metal stress. A reduced APX activity was observed in many crops under heavy metal stress (Smeets et al. 2008; Khan et al. 2009; Pinto et al. 2009).

In halophytic plant K. virginica, Han et al. (2013) reported a 35% reduction in APX activity with Cd (5 μM), and NaCl alone had no effects. Combined effect of both stress (Cd+NaCl) resulted in a significant reduction in APX activity (49%). In S. maritima, the APX activity decreased with salinity but remained the same as in control with heavy metal (As) treatment (Panda et al. 2017). Similarly Bankaji et al. (2016) reported a reduction of 64.3% and 21.4% in APX activity in leaves of Atriplex halimus with the treatment of either Cd2+ or Cu2+ (400 μM). A decrease in APX activity was observed when Cd and NaCl (200 mM) were applied simultaneously to the growing medium as compared to their individual application. Similar responses were observed with Cu2+ in place of Cd2+ as heavy metal. This decrease could be the result of damage caused by metal ion-induced ROS (Sai Kachout et al. 2009). Salinity- and As stress-induced H2O2 is reduced by APX scavenging thus to maintain the appropriate level of H2O2 to be used as signalling molecule. H2O2 is efficiently scavenged by the action of APX as observed by Panda et al. (2017) in halophyte S. maritima. They reported a negative correlation between APX activity and H2O2 concentration. Eyidogan and Oz (2007) suggested that the enhancement in APX activity under stress may be modulated by the overproduction of H2O2 under CAT deactivation.

6.3.5 Glutathione Peroxidase

Glutathione peroxidase GPX (EC 1.11.1.9) is an antioxidative enzyme that catalyses the reduction of hydrogen peroxide, using the glutathione (GSH) pool and thus protecting cells against oxidative damage (Helliwell and Gutteridge 1986). Superoxide radicals are converted to H2O2 by SOD (Dixit et al. 2001), and glutathione antioxidant acts as one of the important protective mechanisms (Bela et al. 2015). Cartegnato et al. (2008) reported a 230% and 300% increase in GPX activity when exposed to 100 μg ml−1 and 800 μg ml−1 Zn showing a positive correlation between GPX activity and Zn concentration in leaves of halophyte A. Marina. Wu et al. (2013) reported an increase in GPX activity in above ground part of halophyte Suaeda salsa seedling grown in Zn (100 μg L−1), Pb (20 μg L−1) or the combination of both metals. Similar results were observed by Mesnoua et al. (2016) in Atriplex halimus L. under Cd toxicity. Yildiztugay et al. (2014) observed increase in GPX activity in response to salinity stress (250–1500 mM NaCl) at 15 days in halophytic plants Salsola crassa showing that it contributes to tolerance mechanism under salinity-induced damage. On the other hand, Haluskova et al. (2009) observed only a slight increase in GPX activity with Pb, Ni and Zn and no effect with Co.

However, a decline of 75.8% in GPX activity under Cd2+ stress (400 μM) was observed in leaves of A. halimus (Bankaji et al. 2016). A decrease in GPX activity was also observed when Cd and NaCl (200 mM) were applied simultaneously to the growing medium as compared to individual treatment. Cu2+ (400 μM) treatment however resulted in a significant increase in GPX activity in NaCl plus Cu2+ as compared to only Cu2+ and only NaCl treatment. Increase in GPX activity may be due to the reason Cu acts as catalyst in the formation of ROS (via Fenton reaction) and causes GSH depletion. The other heavy metal like Cd does not produce ROS directly, but it increases lipid peroxidation and GSH depletion causing an indirect ROS production. Sai Kachout et al. (2009) reported that these ROS cause oxidative damage and alter the antioxidative enzymes activities. GPX may act as a secondary line of stress defence under Zn and NaCl treatments.

6.3.6 Glutathione Reductase

Glutathione reductase (GR) (EC 1.6.4.1) is an important enzyme of ascorbate-glutathione cycle. The monodehydroascorbate formed in the APX reaction can be regenerated to ascorbate by reduced GSH, which is oxidized to form glutathione disulphide (GSSG). For many cellular functions, reduced form of glutathione is required. The reduction of oxidized glutathione to reduced glutathione occurs via the action of GR enzyme in a NADPH-dependent manner (Noctor et al. 2012). Glutathione reductase plays a crucial role in providing protection against oxidative stress in plants by maintaining the endogenous pool of GSH. According to Pilon-Smits et al. (2000), this enzyme has very important function in heavy metal tolerance. Rodriguez-Serrano et al. (2006) reported enhancement in expression of GR coding genes including both transcriptional and post-transcriptional impacts under Cd stress. Han et al. (2013) reported an increase of about twofold in GR activity in Cd-treated plants; however NaCl alone treatment resulted in no significant changes in enzyme activities in K. virginica. However a 2.4-fold increase as compared to control plants was observed in GR activity in plants treated with both Cd and NaCl indicating a better tolerance to heavy metal in presence of NaCl. Lefevre et al. (2010) reported enhancement in GR activity as major component involved in heavy metal (Cd) tolerance in xero-halophyte A. halimus. GR may be involved in the regeneration of reduced glutathione by reduction of oxidized glutathione under salinity stress to maintain redox status of the cells as observed in halophytes. The GR activity gradually decreased in S. maritima with increasing salinity and increased with increasing As level. However when salinity and As treatment both were applied simultaneously, there was no significant increase in GR activity as compared to control plants (Panda et al. 2017). It is an important enzyme and plays an important role in maintaining of redox status in ascorbate-glutathione cycle in halophytes under heavy metal stress and endogenous H2O2 content through an oxido-reduction cycle (Halliwell-Asada pathway) (Bose et al. 2014; Rangani et al. 2016).

6.3.7 Dehydroascorbate Reductase and Monodehydroascorbate Reductase

Monodehydroascorbate reductase (MDHAR) (EC 1.6.5.4) and dehydroascorbate reductase DHAR (EC 1.8.5.1) are also important enzymes of the ascorbate-glutathione pathway. MDHAR is FAD enzyme that catalyses the reduction of monodehydroascorbate radical (Lunde et al. 2006). With its ability to directly regenerate ASA, MDHAR contributes towards maintaining a reduced pool of ASA (Sharma et al. 2012). MDHAR directly uses NADPH to recycle ascorbate. However MDHAR is an efficient electron acceptor (Noctor and Foyer 1998; Asada 2000), and it can be reduced directly to ascorbate using photosynthetic ETC electrons. This enzyme is mainly located in mitochondria and peroxisomes, and along with APX it is involved in scavenging of hydrogen peroxide (Del-Rio et al. 2002; Mittler 2002). Its isozymes are also found in other cellular organelles like chloroplast, mitochondria, cytosol, peroxisomes and glyoxysomes (Hossain et al. 1984; Jimenez et al. 1997; Sharma et al. 2012; Das and Roychoudhury 2014). DHAR like MDHAR also helps in regeneration of ascorbic acid (AA) pool in plant cells (Das and Roychoudhury 2014). Ascorbic acid (AA) is a major antioxidant in plants for ROS detoxification and photosynthetic functioning. The DHAR function as an important regulator of AA recycling by catalysing the regeneration of ascorbic acid from its oxidized form. The univalent oxidation of ascorbic acid leads to the formation of MDHAR, which is converted to DHA via further oxidation reduction. This DHA is then reduced to AA by the action of DHAR also requiring GSH (Eltayeb et al. 2007). Few studies have been conducted on MDHAR and DHAR response under heavy metal stress in halophytes. The overexpression of DHAR genes enhanced the tolerance to environmental stress in tobacco and Arabidopsis (Chen and Gallie 2006; Eltayeb et al. 2007). The increased activity of DHAR under stress conditions may be because of increasing level of AsA. High level of AsA under salinity and As treatment may be one strategy to reduce oxidative damage in S. maritima (Panda et al. 2017). In halophyte K. virginica, DHAR activities increase to about 2.5-fold in Cd-treated plants; however no significant effect was observed under NaCl alone and a combination of both Cd and NaCl (Han et al. 2013). However a 15% reduction in MDHAR activity was observed with Cd stress as compared to control, and no significant effects were observed with both Cd and NaCl and alone NaCl-treated plants.

6.3.8 Non-enzymatic Antioxidants

The non-enzymatic antioxidants include ascorbic acid, glutathione, tocopherols, carotenoids, flavonoids and proline. These antioxidants provide protection to cellular components from damage either by interrupting cascade of uncontrolled oxidation (Noctor and Foyer 1998) or donating an electron to neutralize free radicals to non-reactive species (Oztetik 2012). Ascorbate and glutathione are two important antioxidants involved in ROS detoxification via ascorbate-glutathione cycle under stress conditions (Foyer and Noctor 2005). They are involved in detoxification of ROS-induced oxidative stress. Glutathione will be discussed in details under sulphur metabolism and proline under osmoprotectants subheadings.

Ascorbic acid is an abundant, low molecular weight, water-soluble antioxidant involved in ROS detoxification under stress conditions. It has the ability to donate electrons in a number of enzymatic and non-enzymatic reactions and thus is involved in ROS scavenging (Smirnoff 2005; Gill and Tuteja 2010). It is located in many plant tissues like meristematic and photosynthetic cells. It plays very important role in many processes like plant growth, differentiation and metabolism. In plant cell AA is mainly found in cytosol, with a significant amount in apoplast, and acts as a first line of defence for ROS detoxification (Barnes et al. 2002). The oxidation of AA involves two steps, first to produce MDHAR which, if not reduced immediately to ascorbate, disproportionate to AA and DHA. Ascorbic acid reacts with free radicals H2O2, OH− and O2 − to protect membranes from oxidative damage and also regenerate α-tocopherol from tocopheroxyl radical (Shao et al. 2005). Ascorbic acid also preserves the activities of metal-binding enzymes (Zaefyzadeh et al. 2009).

In halophyte K. virginica, a significant increase of twofold in AsA level was noticed with Cd+NaCl-treated plants as compared to plants exposed to Cd stress alone. However NaCl treatments resulted in no significant change in ascorbate content as compared to combined treatments (Han et al. 2013). Panda et al. (2017) studied the ascorbate content in S. maritima under NaCl, As and combined treatment and reported that the AsA/DHA ratio increased by approximately threefold with salinity treatment of 200 and 600 mM as compared to control plants. With 200 μM As treatment, an 11.8-fold increase in AsA/DHA ratio was observed, but a slight increase was observed with the combined treatment of As (200 μM) and NaCl (200 mM). The increase in ratio is due to either decrease in DHA or increase in AsA content, and the possible reason behind this may be decreased rate of oxidation of AsA and increased rate of its synthesis under stress conditions. The decrease in DHA content under salinity and heavy metal stress may be due to induction in DHAR activity in stress condition or increase in GSH level which acts as electron transport (Foyer and Noctor 2005; Ghosh et al. 2016). In contrast to these results, Demir et al. (2013) reported an increase in DHA levels under combined stress of arsenic and salinity in Cakile maritima. The AsA/DHA and GSH/GSSG ratios are very important for maintaining cellular redox status, and changes in these ratios may be considered as first sign of oxidative stress in plants (Foyer and Noctor 2005). The cellular redox homeostasis helps in detoxification of oxidative stress (Foyer and Noctor 2005; Parida and Jha 2010).

Tocopherols are a group of lipophilic antioxidants collectively termed as vitamin E. Tocopherols have four isomers α, β, γ and δ. The α-tocopherol isomer has maximum antioxidative properties and capability to scavenge ROS and lipid free radicals. These characteristics make it an indispensible protector and essential component of plant biomembranes (Hollander-Czytko et al. 2005; Kiffin et al. 2006). Tocopherols are synthesized in organisms with the ability of photosynthesis, and these are present only in green tissues of plants. Tocopherol uses homogentisic acid (HGA) and phytyl diphosphate (PDP) as precursors for its biosynthesis. The main enzymes involved in the biosynthesis of tocopherol are 4- hydroxyphenylpyruvate dioxygenase (HPPD), homogentisate phytyl transferases (VTE2), 2-methyl-6-phytylbenzoquinol methyltransferase (VTE3), tocopherol cyclase (VTE1) and γ-tocopherol methyltransferase (VTE4) (Li et al. 2010). The major role of tocopherol is protection of PSII. They can quench excess energy of PSII by reacting with O2 and thus protect the lipids and other membrane constituents of chloroplast. They serve as a potent free radical scavenger due to ability to inhibit chain propagation step of lipid peroxidation cycle. α-Tocopherol can scavenge free radicals like RO., and ROO present in the membranes convert them into TOH• which interacts with GSH and AA and is subsequently recycled to its reduced form (Igamberdiev et al. 2004). Oxidative stress has been reported to activate the expression of tocopherol biosynthetic genes in higher plants (Wu et al. 2007). Han et al. (2013) reported decrease in levels of α-tocopherol (35%) under Cd (5 μM) treatment in response to Cd-induced oxidative stress in halophytic plant K. virginica. Only 50 mM NaCl in nutrient solution slightly increased the α-tocopherol levels as compared to control, but an increase of about 28% was observed with NaCl in Cd-treated plants showing the enhanced protective mechanism in halophytes.

Carotenoids are lipophilic non-enzymatic antioxidant pigments. They can be synthesized under stress conditions and have the ability to scavenge and deactivate ROS (Safafar et al. 2015). These antioxidative pigments are also found in plants as well microorganisms. They further include two types of pigments xanthophylls (contain oxygen) and carotenes (purely hydrocarbons and contain no oxygen). Carotenoids are present in chloroplast membranes. They quench singlet oxygen which is generated when light energy absorbed by chlorophyll is not dissipated through photosynthesis and protect chlorophyll from photooxidative damage. Therefore, a reduction in carotenoids has a serious consequence on chlorophyll pigments. They react with lipid peroxidation products to terminate chain reactions, thus stopping further damage. They inhibit oxidative damage and quench triple sensitizer (3Chl*), excited chlorophyll molecules (Chl*) thus preventing the accumulation of singlet oxygen and protect photosynthetic machinery from further damage (Dar et al. 2017).

da Silva (2010) reported an overall increase in chlorophyll a, chlorophyll b and carotenoid content up to 400 μM Zn treatments, and above it Zn was found toxic for the H. portulacoides plants. Bertrand and Poirier (2005) stated that many studies showed decrease in photosynthetic pigment under heavy metal stress, but some other researchers observed an increase in carotenoid content (Mallick and Rai 1999; Mallick 2004). MacFarlane and Burchett (2001) reported a significant decrease in carotenoid content when exposed to Cu (800 μM) treatment in a mangrove Avicennia marina. Pb treatment showed no significant effect on carotenoid content, but decline was observed with Zn at 1000 μg/g in the growing medium. Cu and Zn are mobile but show restricted translocation to shoot due to endocasparian strips, while Pb is actively excluded at root, thus having less effects on plant metabolism. Carotenoid contents of S. maritima did not vary significantly under arsenic, salinity and combined treatments (Panda et al. 2017). No effects on chlorophyll content under salinity and heavy metal stress may be due to no effect on chlorophyllase activity, thereby protecting chlorophyll degradation (Rangani et al. 2016). Sghaier et al. (2015) studied the Tamarix gallica under salinity, As and salinity+arsenic treatment and reported no change in level of photosynthetic pigments, chl a, chl b, chl a/b, total chlorophyll and carotenoids. In contrast salinity-induced decline in carotenoid content was observed in other halophytes like Arthrocnemum macrostachyum (Redondo-Gomez et al. 2010); chenopodiaceous halophytes including Suaeda salsa (L.), Kochia scoparia (L.) and Beta vulgaris (L.) (Takagi and Yamada 2013); Quinoa (Amjad et al. 2015); and Panicum turgidum (Koyro et al. 2013).

Flavonoids are mainly found in plant leaves, floral parts and pollens. They act as important secondary antioxidants by scavenging ROS, locating and neutralizing radicals before cell damage and protecting photosynthetic damage under environmental stress conditions (Fini et al. 2011).

6.4 Sulphur Metabolism and Its Role in Heavy Metal Detoxification

Sulphur (S) is an ubiquitous and essential macronutrient in plants. In higher plants its uptake is mainly in the form of sulphate anions from the soil which through a series of reactions is converted into organic S-containing compounds like the amino acids, cysteine (Cys) and methionine (Met). These organic compounds are essential components of many proteins, lipids, polysaccharides, iron-sulphur cluster, many vitamins (biotin and thiamine), cofactors (CoA and S-adenosyl-met), some peptides (glutathione, phytochelatins and metallothioneins), etc. In addition some amount of sulphur can also be taken up in the form of sulphur dioxide (SO2) and hydrogen sulphide from the air which again through a cascade of reactions gets converted to organic S-containing compounds. Sulphur-containing compounds are involved in many biochemical and physiological processes via regulation of enzymatic activities and redox balance including abiotic stress tolerance mechanism particularly in heavy metal detoxification (Gill and Tuteja 2010). The sulfhydryl group of cysteine residues has high nucleophilicity which makes them react with a number of molecules like free radicals, reactive oxygen species, toxic electrophilic xenobiotics and heavy metals (Leustek et al. 2000; Nocito et al. 2007). These effects indicate a unique role of cysteine in biological systems. Aslund and Beckwith (1999) explained that the two cysteine residues of a polypeptide chain may interact in an oxidation reaction through the formation of a reversible double bond which helps in maintenance of protein structure and regulation of protein activity. This interconversion of two thiol groups in disulphide bond may involve balance of redox cycles. In redox cycles balance transfer of electron is a must during normal and oxidative stress conditions. The sulphur-containing molecules play an important role in heavy metal detoxification in halophytes also. The important sulphur-containing molecules are discussed below.

6.4.1 Glutathione

Glutathione is an important low molecular weight thiol in plant cells. It has been detected in almost all cell organelles like cytosol, endoplasmic reticulum, mitochondria, chloroplast and vacuole (Yadav 2010). It is an important and essential metabolite with multiple functions including defence against salinity- and heavy metal-induced ROS production (Foyer and Noctor 2005), heavy metal sequestration (Cobbett and Goldbrough 2002; Freeman et al. 2004) and xenobiotics detoxification (Dixon et al. 1998). Two principal structural features of glutathione (GSH) are presence of thiol group on cysteine and γ-glutamyl linkage. GSH is the major transport and reduced form of sulphur in plants. It is synthesized by two ATP-dependent reactions by the action of glutamylcysteine synthetase catalysing the formation of peptide bond between carboxyl group of glutamate and amino group of cysteine to yield γ-glutamylcysteine (γ-EC). It is the regulatory step under the high/more GHS requiring conditions (Noctor et al. 1998). The second reaction encompasses glutathione synthetase which catalyses the bond formation between glycine residue and γ-glutamylcysteine (γ-EC) to form GSH. According to Wachter et al. (2005) studies, GSH1 is present in the plastids, and GSH2 is present in both plastids and cytosol in case of Arabidopsis thaliana and Brassica juncea. Cysteine is proposed to be the rate-limiting factor for glutathione biosynthesis. Cysteine is the final product of sulphur assimilation pathway, and GSH biosynthesis is correlated with S assimilation pathway (Rausch and Wachter 2005). Glutathione plays a very important role in a number of biological processes, and it is the most abundant intracellular thiol and γ-glutamyl compound. Further its physiological significance can be divided into two categories. First one is as an important pool for reduced sulphur and regulation of sulphur uptake by plant roots, and second its cysteine residue helps in chelation and thus in antioxidative defence system and redox control for salt and heavy metal detoxification in halophytes.

The main basic and recognized function of glutathione is when its reduced form is oxidized to its disulphide form (GSSG) during thiol-disulphide interactions and the GSSG is again recycled to GSH by the action of NADH-dependent glutathione reductase enzyme in cell organelles and cytosol where it is present. This recycling is rapid, and the simple cycle represents the basis for confirming its role as a powerful redox buffer (Gill and Tuteja 2011). During oxidative stress GSH gets oxidized first by ROS as a part of antioxidant defence system and prevents excessive oxidation of other sensitive components of cell. GSH can scavenge ROS such as O2 −, H2O2 and OH− radicals produced as a result of stress (Gill and Tuteja 2011). The features of GSH like its high concentration and its high reduction state make it a favourable antioxidant under stress conditions. Unlike the other antioxidative enzymes and metabolites that scavenge ROS, the oxidized GSSG is rapidly recycled to GSH by GR in cell organelles and cytosol (Halliwell and Foyer 1978; Kataya and Reumann 2010). GSH also protects cell membranes by maintaining zeaxanthin and tocopherol in the reduced state (Gill and Tuteja 2011). This property protects the plant from the deleterious effects of combined salt and heavy metal stress. The thiol group of GSH is responsible for its chemical reactivity and functions. The nucleophilic nature of the thiol group in GSH is important in the formation of mercaptide bond with metals as well as in reacting with some electrophiles (Yadav 2010). These kinds of reactions are catalysed by glutathione S-transferase (GST) and have been reported to be required in the process of detoxification of different abiotic stresses (Marrs 1996; Leustek et al. 2000). Additionally, GSH is a substrate for GPX and GST (Gill and Tuteja 2010). The conjugated forms are then transported to the vacuole and protect the plant cells from harmful toxic effects (Klein et al. 2006). However, the consumption of reduced glutathione in xenobiotic or heavy metal stress results in a transient decrease of cytosolic glutathione (GSH) content. This affects the GSH/GSSG redox potential, inducing a redox signal under stress conditions (Nocito et al. 2006). Therefore, GSH-based detoxification process will have an effect on redox potential of cell, and thus maintenance of GSH/GSSG ratio becomes very essential for the survival of plants in heavy metal stress conditions (Gill and Tuteja 2011). Halophytes generally have a high GSH/GSSG ratio and better tolerance to heavy metal stress. Higher GSH content maintains the thiol group containing proteins and enzymes in their native stage under stress conditions and has been reported to increase the salinity and arsenic tolerance potential in halophytic plant S. maritima (Vromman et al. 2016). Panda et al. (2017) reported that the ratio of reduced and oxidized glutathione (GSH/GSSG) did not change under salinity, heavy metal (As) and their combined stress and this helped in maintaining cellular redox balance in S. maritima. Higher GSH content corresponds to higher tolerance against heavy metal stress-induced oxidative damage (Freeman et al. 2004). It is suggested that production of GSH under stress conditions in halophytic and metal-accumulating plants shared a common mechanism. Increase in GSH content and/or other antioxidative enzymes has been reported in a number of halophytes. Overexpression of S. brachiata glutathione transferase in tobacco conferred tolerance up to 300 mM NaCl (Jha et al. 2011). Similarly, Lokhande et al. (2011) have observed the role of glutathione and ascorbate as antioxidant in balancing cellular redox homeostasis in halophyte S. portulacastrum (L.) under salinity stress. Therefore, manipulating GSH biosynthesis by genetic engineering in plants under stress conditions results in increased tolerance to ROS (Sirko et al. 2004). In vitro studies with the biosynthetic enzymes from parsley and tobacco reported a feedback inhibition of γ-glutamylcysteine synthetase (γ-ECS) by reduced glutathione (GSH) (Noctor and Foyer 1998). Exogenously applied GSH had a positive effect on the indices of stress under metal treatments (Cai et al. 2010). Limited information is available regarding transport of GSH-metal complex across the membranes; however, an ATP-binding cassette transporter AtATM3 has been found as GSH-Cd transporter across mitochondrial membrane. This transporter was isolated from the mitochondrial protein of A. thaliana. Kim et al. (2006) reported the induction of AtATM3 gene expression in plant roots in response to Cd and Pb stress, indicating the role of ATP-binding cassette transporter in the regulation of cellular GSH levels and thus oxidative metabolism.

Reduced glutathione is a direct precursor for the synthesis of phytochelatins, a Cys-rich peptide involved in heavy metal detoxification (Zenk 1996). Low GSH content has been correlated with Cd sensitivity, and this may be due to less PC synthesis (Xiang et al. 2001).

6.4.2 Phytochelatins (PCs)

PCs have been reported to be present in the plant kingdom including all angiosperms, gymnosperms and bryophytes (Gekeler et al. 1989). These were first isolated from plant cell suspension cultures upon Cd exposure (Grill et al. 1985). Besides Cd, other heavy metals like Cu, Zn, Pb, Ni and Hg have been reported to induce the synthesis of PCs. Generally plants enhance the synthesis of phytochelatins in heavy metal stress conditions (Sruthi et al. 2017). PCs contain sulphur which plays an important role in their synthesis and also in detoxification of heavy metals by forming heavy metal-binding peptides particularly Cd-binding peptides (CdBP) (Cobbett and Goldsbrough 2002). PC synthesis in response to Pb treatments leads to the formation of PC-Pb complex (Piechalak et al. 2002). PC translocation studies in Arabidopsis have shown that they can transport from roots to shoots through a long-distance transport mechanism. Studies with xylem and phloem sap from Brassica napus have confirmed the transport of PCs. Further it was reported that PCs and Cd are present in high concentrations in the phloem sap as compared to xylem sap, thus indicating that PCs can function as long-distance carriers of Cd (Yadav 2010). Cd mainly forms PC-Cd and GSH-Cd complexes and gets transported (Mendoza-Cozatl et al. 2008). Phytochelatins have a role in heavy metal detoxification and in maintaining ion homeostasis (Zenk 1996; Hirata et al. 2005). Though hyperaccumulation does not use this expensive strategy commonly to cope with high doses of heavy metals, the presence of constitutive and functional PCs are ancestral characters (Petraglia et al. 2014) that may help the plant to minimize damage under conditions of excess metal concentration in non-metallophytes (Tennstedt et al. 2009). Now a lot of database is present in public sector after the cloning of PCS gene. Expressed sequence tag (EST) data supports the view that PCS genes are present in all higher plants. Sequences homologous to PCS have been reported in various monocot and dicot plants. Yadav (2010) also reported sequence data for the presence of PCS genes in ferns and diatoms. The role of phytochelatins in regulation of heavy metal stress in nodulated plant such as Lotus japonicus has also been reported (Ramos et al. 2007, 2008).

Phytochelatins have a general structure (γ Glu-Cys)nGly, where n varies from 2 to 11. These are enzymatically synthesized polypeptides using glutathione as precursor in the presence of enzyme phytochelatin synthase (PCS) and help in metal detoxification by forming complex with metal-Cd complex and transporting it to the vacuole (Sachiko et al. 2009; Gill and Tuteja 2011). The sulfhydryl group of PC forms a complex with heavy metal as reported for arsenic in rice (Tripathi et al. 2012) and xero-halophyte Atriplex atacamensis (Vromman et al. 2016). They reported that arsenic is converted to arsenite and binds to SH group of PCs and forms a PC-As complex which then is transported to vacuole where pH range favours the high molecular weight PC-As complexes. However sequestration of the PC-Pb complex to vacuole has not been reported (Yadav 2010). The PCS has been reported to be activated on treatment with heavy metals like Cd (Cobbett and Goldbrough 2002; Thangavel et al. 2007). The halophytic plant black mangrove Avicennia germinans exhibits tolerance to polluted environment and is correlated with overexpression of AvPCS coding genes induced by heavy metals Cd and Cu during initial hours of treatment (Gonzalez-Mendoza et al. 2007). This overexpression is transient and is sufficient to trigger an efficient detoxification mechanism against long-term heavy metal stress. Heavy metal-induced oversynthesis of PCs in halophytes contributing towards the tolerance mechanisms may be able to increase glutathione concentration in a better way than glycophytes. The intracellular level of GSH determines the level of PCs that a plant is able to synthesize under stress conditions (Liu et al. 2015). However Panda et al. (2017) observed that a higher GSH content under As treatments did not reflect in higher PC synthesis in S. maritima which could be due to As-PC complex formed in roots, lesser transport of As from root to shoot. Phytochelatins are involved in detoxification of a wide range of heavy metal ions like Cd2+ and Cu2+. Bankaji et al. (2016) demonstrated that Cd2+ and Cu2+ stress caused PC accumulation in the halophyte Atriplex halimus that can be considered as hyperaccumulating species.

High GSH levels activate PCS which catalyses PC-heavy metal complex. The enzyme PCS become active upon the formation of a thiolate between two GSH molecules and heavy metals (Cd-GS2)/(Zn-GS2). Vatamaniuk et al. (2000) reported that this activation also involves the transfer of γ Glu-Cys moiety to free GSH or already synthesized PC. These PC-heavy metal complexes are transported to vacuole, and this is a more stable form of heavy metals (Mendoza-Cozatl et al. 2005)

Manipulation of PCS by genetic engineering and mutant studies has further confirmed the role of PC in heavy metal tolerance (Yadav 2010). Pomponi et al. (2006) reported that availability of GSH is directly correlated with overexpression of AtPCS1 genes for Cd tolerance and accumulation. The heavy metals Pb, Cu, Zn and Cd resulted in overexpression of TaPCS1 (a wheat PCS) in shoots of plants when grown in polluted soil (Gisbert et al. 2003; Martinez et al. 2006). However, in some plants increase in PC synthesis was observed without stimulated GSH that leads to GSH reduction and plant found less tolerant to heavy metal contaminants. Zhu et al. (1999) reported an increased tolerance to Cd in overexpressed γ-Glu-Cys synthase gene from E. coli in Brassica napus. Lee and Kim (2010) reported the expression of Arabidopsis PCS in a yeast mutant deficient in PCS resulted in tolerance to Cd but not to Cu. Sachiko et al. (2009) revealed in mutation studies in PC synthase that replacing Cys residue with Ala resulted in less synthesis of PC; furthermore the mutant PCS was sensitive to oxidative conditions. This work emphasized on the importance of Cys-rich region in PCS for antioxidative activity.

6.4.3 Metallothioneins (MTs)

MTs are also cysteine-containing proteins which bind with metals and are found in almost all organism including plants, animals and microorganisms. Based on the location of cysteine residue, plant MTs have been divided into four types (Cobbett and Goldbrough 2002). These proteins were originally identified in animals for their ability to protect against Cd toxicity. Besides metal chelation MTs have been proposed to be involved in other cellular processes also like regulation of cell growth and proliferation, repair of DNA damage and scavenging of reactive oxygen species (Cherian and Kang 2006). MT superfamily is comprised of low molecular weight polypeptides generally less than 10 K Da, high metal content, low aromatic amino acid content and amino acid sequence motifs containing numerous Cys residues with characteristic distribution and spectroscopic expressions typical of metal thiolate clusters (Freisinger 2011). MTs bind to different heavy metals by formation of mercaptide bonds between its cysteine residue and metal. Therefore the arrangement of cysteine residues is important in determining the metal-binding properties of MTs (Cobbett and Goldbrough 2002; Blindauer and Leszczyszyn 2010). On the basis of cysteine amino acids arrangement, the plant metallothioneins have been grouped into four types: MT1, MT2, MT3 and MT4. Pagani et al. (2012) reported the expression of MTs in soya bean. GmT1, GmT2 and GmT3 were ubiquitously expressed and GmT4 synthesis was restricted to seeds. GmT1 is mainly present in roots; GmT2 and GmT3 are predominant in leaves and GmT4 in the seeds. GmT2 and GmT3 were highly responsive to Cd treatments indicating their role in Cd detoxification mechanism. MT Cd binding has been reported in A. thaliana (Zimeri et al. 2005) and Vicia faba (Lee et al. 2004).

Guo et al. (2008) studied the expression of Arabidopsis MTs in the copper-sensitive yeast mutant D cup1 and found tolerance and accumulation equal to wild-type leaves. This copper tolerance indicates MTs are able to function as Cu chelators. Expression of all four MTs provided increased tolerance to some extent in Zn-sensitive mutants also; however MT4 which is expressed in the seeds provided higher Zn tolerance and accumulation.

These MTs are also involved in balancing metal homeostasis or tolerance mechanism as they can also bind metals. Studies conducted on Bruguiera gymnorrhiza (L.) Lam., a most salt-tolerant mangrove halophyte, showed that the genes coding for MT2 (type −2) were upregulated by metals Zn, Cu and Pb (Huang and Wang 2009). Further (Huang and Wang 2010) reported higher tolerance to Zn, Cu, Pb and Cd in a halophytic mangrove Avicennia marina where transgenic E. coli LB21 overexpressing a gene coding for the protein GST-AmMT2. Location wise MT2 was mainly present in leaves while MT1 was in roots of plants. Salicornia brachiata is an extreme halophyte generally exposed to heavy metals in coastal areas. Chaturvedi et al. (2014) transformed tobacco with Salicornia brachiata MT2 gene for its functional validation. SbMt2 was found to be involved in binding and accumulation of Cu and Cd. They also reported that transgenic lines overexpressing SbMT2 genes showed a high ratio under stress conditions thus indicating that SbMT2 plays a role in ionic homeostasis also. Thus it may be hypothesized that overexpression of the genes coding for MT2 in halophytic plants shows positive function in relation to tolerance to heavy metals. However different elements have different impact on MT2 coding genes. Similarly Usha et al. (2009) reported that significant upregulation of PjMT2 in Prosopis juliflora with Zn and Cu and Cd treatment did not change the expression. Similarly in Salicornia brachiata, SbMT-2 expression was upregulated by Zn and Cu but not affected by Cd metal (Chaturvedi et al. 2012). The sequestering ability of MTs depends on the metal also as observed in Salicornia brachiata a fourfold higher Zn sequestration was observed as compared with Cu.

Role of MTs genes in antioxidant defence responses has also been reported by many researchers in the literature (Yang et al. 2009; Wang et al. 2009; Xue et al. 2009; Zhu et al. 2010; Kumar et al. 2013; Chaturvedi et al. 2012, 2014). Chaturvedi et al. (2012) have also observed that SbMT-2 expression from the halophyte may be enhanced by salinity, drought and heat, but a decreasing effect was found with cold stress. Further studies by Chaturvedi et al. (2014) showed that SbMT2 gene expression in tobacco mitigates the deleterious effect of salt, drought and metal stress. Lower membrane injury, lipid peroxidation, H2O2 and proline content and high activity of antioxidative enzymes like SOD, POX and APX transgenic lines indicate the possible role of SbMT2 in ROS scavenging and detoxification mechanism under stress conditions.

6.5 Osmoprotectants

Low molecular weight organic compounds generally called compatible solutes/osmolytes are water soluble, independent (to metabolic reactions) and non-toxic in nature even at higher cellular concentrations.

Strong resistance of halophytes to heavy metals tightly shows its parallel characteristics of salt resistance. Increase in concentration of osmoprotectants helps halophytes to fight against heavy metals and other abiotic stresses by maintaining water balance through lowering of osmotic potential, thus making all the physiological functions happening normally (Flowers and Colmer 2008). Metal stress creates water stress which in turn causes oxidative damage to cellular structures, and the concentration of osmoprotectants is directly related to heavy metal tolerance, giving clear indication of the role of halophytes in phytoremediation of heavy metals in saline patches. The speed of synthesis of osmolytes is also directly correlated with degree of ion toxicity and thus explains their potential of phytoremediation.

Different osmolytes synthesized by plants have been identified and listed in Fig. 6.1, including different polysaccharides (trehalose, glucose, sucrose, raffinose fructose and fructans); sugar alcohols (polyols) like sorbitol, mannitol, inositol and methylated inositols; amino acids, such as methyl-proline, proline betaine, proline, pipecolic acid and hydroxyproline betaine, glycinebetaine and choline O-sulphate; and tertiary sulphonium compounds, such as dimethylsulphoniopropionate (DMSP) (Rhodes et al. 2002; Ashraf and Foolad 2007).

Fig. 6.1
figure 1

Structures of common osmolytes present in halophytes

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6.5.1 Proline

In combating oxidative damage, plants face numerous challenges, and to overcome the stress, they explicit different strategies, one of which is the synthesis of osmolytes which provide protection against the damage caused to cellular structures and making the plants survive under stress conditions (Parida and Das 2005). One such osmolyte is proline, for which the precursor is glutamate; the enzyme catalysing the reaction is pyrroline-5-carboxylate synthetase (P5CS) (a key enzyme in proline biosynthesis) to pyrroline-5-carboxylate (P5C) (Szabados and Savoure 2010) which further reduces to proline. Mitochondria are the site of proline catabolism, which is catalysed by the enzyme proline dehydrogenase or proline oxidase (ProDH) to glutamate (Servet et al. 2012). The second precursor for proline biosynthesis is ornithine, which on transamination produces proline via enzyme ornithine-d-aminotransferase (Verbruggen and Hermans 2008).

The action of proline differs at different levels and in different tissues. It is involved in the alleviation of cytoplasmic acidosis and sustaining NADP+/NADPH ratios required for metabolism (Hare and Cress 1997). Proline accumulates under stress conditions and serves as a sink for excess reductants, providing the NAD+ and NADP+ necessary for maintenance of respiratory and photosynthetic processes.

Commonly proline gets accumulated in cytosol, parallelly with vacuolar Na+ sequestration. The cell membrane integrity is maintained by the interaction of phospholipids with proline, which in turn also keeps the quaternary structures of proteins intact. Sharma and Dietz (2006) and Lefevre et al. (2009) have observed three major roles of proline under heavy metal stress, viz. metal binding, antioxidant defence and signalling. Chai et al. (2013) observed the mixed effects of NaCl on phytotoxicity caused by Cd stress in relation to proline accumulation. Lefevre et al. (2009) and Saiyood et al. (2012) reported that Suaeda maritima showed that resistance to mixture of inorganic pollutants was directly proportional to its capability to add proline and soluble sugars in the roots, stems and leaves with similar finding in Atriplex halimus, in response to Cd treatment.

Siripornadulsil et al. (2002) showed the positive relation between the increased proline levels and improved protection against Cd, in microalgae. The possible reason is that proline reduces Cd-induced free radical damage and maintains a stringent reducing environment (higher GSH levels) within the cell. Huang et al. (2010) explained that proline, GSH and PCs-SH play an important role in ameliorating the effect of HM toxicity in two mangroves Kandelia candel and Bruguiera gymnorrhiza exposed to multiple HMs (Cd2+, Pb2+ and Hg2+) by maintaining optimum metabolism (physiological and biochemical). Heavy metal induces both a secondary water stress (Nedjimi and Daoud 2009) and oxidative stress in plants (Verma and Dubey 2001). In addition to its role as an osmolyte for water economy, proline helps to stabilize subcellular structures (e.g. membranes and proteins), scavenge free radicals and buffer cellular redox potential under stress conditions (Ashraf and Orooj 2006). The proline content basically maintains the balance between the cytoplasmic water potential to that of vacuole, where most of the inorganic ions are sequestered (Flowers et al. 1986).

Thomas et al. (1998) reported that in leaves of M. crystallinum, a tenfold increase in copper caused 15-fold increase in the proline. However, the magnitude of increase was more when the plants were exposed to salt stress. Proline accumulates in metal-stressed plant cells even when they are turgid, along with their additional role as an antioxidant/chelator (Sharma and Dietz 2006).

From the above discussion, it seems that halophytes can better cope with heavy metal stress than other plants as they have the ability to absorb them from soil and then degrade or reduce them to their organic forms which are non-toxic as referred in Table 6.2.

Table 6.2 Effect of heavy metal stress on osmoprotectant levels in different halophytes
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6.5.2 Glycinebetaine

Glycinebetaine is also one of the compatible solutes found in the cytoplasm in salt-tolerant plants. Choline is the precursor of glycinebetaine synthesis, which is converted to betaine aldehyde and to glycinebetaine through a series of enzymatic reactions of choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. The other pathways known are the direct N-methylation of glycine, but choline pathway is the main biosynthetic pathway present in all plant species (Ashraf and Foolad 2007; Fitzgerald et al. 2009).

Halophytic plant species shows the triggered synthesis of glycinebetaine on exposure to high concentration of heavy metals and NaCl. The increased concentration of glycinebetaine serves as an intercellular osmoticum, and the increase corresponds to rise of osmotic pressure and helps in maintaining membrane integrity by protection of cellular structures, viz. chloroplasts and photosystem II. Bose et al. (2014) reported superior ability of halophytes to accumulate glycinebetaine. Han et al. (2013) reported that accumulation of glycinebetaine in response to higher amounts of NaCl delays Cd-induced senescence, possibly by the maintenance of maximum quantum yield efficiency, effective quantum yield of photosystem II and electron transport rates. Transgenic plants having transgenes for glycinebetaine or proline have not been accepted as they could not produce the required amount of glycinebetaine or proline that can ameliorate the abiotic stresses. Alternatively Ashraf and Foolad (2007) demonstrated that external application of GB or proline to plants under stress has significant effects. Lefevre et al. (2009) reported oversynthesis of glycinebetaine in leaves of Atriplex halimus under Cd stress, whereas in Kosteletzkya virginica, copper stress exhibited the same results (Han et al. 2012).

Sesuvium portulacastrum is a well-known metallotolerant halophyte and also accumulates trans-4-hydroxyprolinebetaine and 3,5,4-trihydroxy-6,7-dimethoxyflavone 3-glucoside. In contrast the concentration of glycinebetaine decreased in Suaeda salsa, in response to Cd stress with increase in choline (Liu et al. 2008). As betaine is synthesized from choline, the authors suggested that the elevated choline should be related to the depleted glycinebetaine.

6.5.3 Soluble Sugars

In glycophytes osmotic potential is maintained to the tune of 50% by soluble sugars under saline conditions. After understanding the physiological mechanism of salt tolerance in halophytes, it was found that there is significant contribution and higher accumulation of soluble carbohydrates under salinity stress (Kerepesi and Galiba 2000; Parida et al. 2002).

Polyols also play a key role in adapting halophytes to abiotic stress, by compensating the reduced cell water potential, and as oxygen radical scavengers; maintain enzyme activities; and protect membrane structures by their hydroxyl groups, in the case of cellular dehydration (Noiraud et al. 2001). Thus, apart from their established functions as precursors of metabolic compounds, they also act as signalling molecules and major cellular energy source under abiotic stresses (Gupta and Kaur 2005; Hare et al. 1998). Parida and Das (2005) stated that carbohydrates act as osmoprotectants ROS scavengers, maintain osmotic balance and serve as molecules for carbon storage under stress conditions.

Sucrose and trehalose sugars accumulate in plants in response to abiotic stresses (Yuanyuan et al. 2009), acting as osmoprotectants, and membranes stabilization (Lokhande and Suprasanna 2012), along with control of several other important metabolic activities. Sugars help to sustain the growth of sink tissues, controlling the expression of several genes either positively or negatively, involved in photosynthesis, respiration and the synthesis and degradation of starch and sucrose (Hare et al. 1998). Trehalose, a non-reducing disaccharide, is highly soluble but chemically unreactive, making it compatible with cellular metabolism even at high concentrations. Trehalose is present in significant concentrations in several bacteria and fungi but rare in vascular plants (Fernandez et al. 2010; Lunn et al. 2014). Resurrection plants are first to observed for trehalose accumulation upon desiccation.

Chai et al. (2013) reported that NaCl reduced soluble sugar content under moderate Cd stress; the possible reason may be that some soluble sugars were used to synthesize proline by providing carbon skeleton and energy (Manuel and Reigosa 2001). However, the slightly reduced levels of soluble sugar under severe Cd stress may be related to the alleviated osmotic stress with addition of NaCl. Thus, the mechanism of soluble sugar in halophytes in response to NaCl under different Cd stresses may be varied. In the plants Syrian beancaper (Zygophyllum fabago) and Atriplex halimus, soluble sugars accumulated in the most of the Cd- and Zn-resistant plants, and also they had higher concentration of soluble sugars than the sensitive plants in response to heavy metal stress (Lefevre et al. 2009).

6.6 Conclusions

Halophytes are expected to be more capable to cope with heavy metal stress than glycophytes. One of the main tolerance mechanisms of halophytes involves oxidative metabolism. Halophytes have inherent salinity-tolerant oxidative metabolism which also confers heavy metal tolerance. The coordination of enzymatic and non-enzymatic antioxidative system in halophytes plays a very important role in providing salinity and heavy metal tolerance. Sulphur metabolism also plays an important role in heavy metal tolerance in halophytic plants via the synthesis of glutathione, phytochelatins and metallothioneins. Glutathione, phytochelatins and metallothioneins contribute towards metal detoxification and compartmentalization in halophytes. GSH maintains the redox potential and contributes towards increased HM tolerance in halophytes and is an important antioxidant metabolite. Minor roles of phytochelatins and metallothionein in ROS scavenging have also been proven. The accumulation of osmoprotectants like proline, glycinebetaine and sugars under heavy metal stress in halophytes results in osmotic protection, thus maintaining water status and nutrient uptake. Halophytes can be suitable candidates for phytoremediation/phytostabilization of metal-contaminated areas due to their high tolerance to heavy metals (Fig. 6.2).

Fig. 6.2
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Effects of oxidative metabolism, sulphur metabolism and osmoprotectants on heavy metal tolerance and detoxification mechanism in halophytes

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