Keywords

8.1 Introduction

Soil salinity is one of the most detrimental abiotic stresses that critically damage crops and cause major reductions in their yield (Munns et al. 2006; Tanveer and Shah 2017). Salinity is characterized by deleterious effects on plant growth, which are traditionally associated with reduced water availability under hyperosmotic saline conditions, and specific ion toxicity. In recent years, oxidative damage has been added to this list (Souza et al. 2012; Liu et al. 2017; López-Gómez et al. 2017). Like other aerobic organisms, higher plants require oxygen for efficient production of energy. During the reduction of O2 to H2O, reactive oxygen species (ROS)—namely, superoxide radicals (O2 −), H2O2, and hydroxyl radicals (OH−)—are formed (Demidchik 2015). Most cellular compartments in higher plants have the potential to become a source of ROS (Bose et al. 2014). Environmental stresses that limit CO2 fixation, including salinity, reduce nicotinamide adenine dinucleotide phosphate (NADP+) regeneration by the Calvin cycle. Consequently, the photosynthetic electron transport chain is over-reduced, producing O2 − and singlet oxygen (1O2) in chloroplasts (Wu and Tang 2004; Shao and Chu 2005). To prevent overreduction of the electron transport chain under conditions that limit CO2 fixation, higher plants have evolved the photorespiratory pathway to regenerate NADP+ (Shao and Chu 2005). As a part of the photorespiratory pathway, H2O2 is produced in the peroxisomes, where it can also be formed during the catabolism of lipids as a by-product of β-oxidation of fatty acids (Foyer and Noctor 2005; Wu et al. 2007).

Because of the highly cytotoxic and reactive nature of ROS, their accumulation in plant tissues and intracellular compartments must be tightly controlled. Higher plant metabolism must be highly regulated in order to allow effective integration of a diverse spectrum of biosynthetic pathways that are reductive in nature (Crawford 2006; Grun et al. 2006). This requires the provision that the regulation does not completely avoid photodynamic or reductive activation of molecular oxygen to produce ROS, particularly superoxide, H2O2, and 1O2 (Suzuki et al. 2012). However, in many cases, the production of ROS is genetically programmed, is induced during the course of development and by environmental fluctuations, and has complex downstream effects on both primary and secondary metabolism (Schurmann 2003; Link 2003; Rouhier et al. 2003). As a result, higher plants possess very efficient enzymatic and nonenzymatic antioxidant defense systems that allow scavenging of ROS and protection of plant cells from oxidative damage (Foyer and Noctor 2003; Anjum et al. 2016a, 2017).

For many years, the concept “the higher the antioxidant activity, the better the plant” has dominated the literature. However, in recent years it has become apparent that plants actively produce ROS as signaling molecules to control numerous physiological processes such as defense responses and cell death (Zhang et al. 2003), cross-tolerance (Bowler and Fluhr 2000), gravitropism (Mittler et al. 2004), stomatal aperture (Wang and Song 2008; Pei et al. 2000), cell expansion and polar growth (Joo et al. 2005; Pedreira et al. 2004), hormone action (Pei et al. 2000; Schopfer et al. 2002), and leaf and flower development (Sagi et al. 2004). In many cases, production of ROS is genetically programmed, and superoxide and H2O2 are used as second messengers (Foyer and Noctor 2005). A new concept of “oxidative signaling”—instead of “oxidative stress”—has been proposed (Foyer and Noctor 2005). The “positive” role of ROS has been reported at both the physiological level [e.g., regulation of ion channel activity (Foreman et al. 2003)] and the genetic level [e.g., control of gene expression (Shin and Schachtman 2004)]. This has prompted a need to rethink the above “the more, the better” concept and incorporate the signaling role of ROS and the redox state of the cell into breeding programs aimed at improving abiotic stress tolerance. Some aspects of this work are discussed in this chapter.

8.2 Antioxidant Defense System

The distinct subcellular localization and biochemical properties of antioxidant enzymes, their differential activation at the enzyme and gene expression level, and the plethora of nonenzymatic scavengers render the antioxidant system a very versatile and flexible unit that can control ROS accumulation temporally and spatially (Shao et al. 2007a, b; Anjum et al. 2015, 2016b). The sections below describe mechanisms involved in redox regulation for salinity stress tolerance in plants.

The redox state of a cell explains the ratio of the amount of oxidizing equivalents to the amount of reducing equivalents (Gabbita et al. 2000). The intracellular antioxidant systems form a powerful reducing buffer, which affects the ability of the cell to counteract the action of the prooxidant forces. The fine redox balance within a cell is thus governed by the levels of prooxidant molecules and antioxidant fluxes. Therefore, an appreciation of the different sources of oxidants and the counteracting antioxidant (or reducing) systems is necessary to understand what factors are involved in achieving a particular intracellular redox state. Salinity induces various ROS and, in response to that, plants develop complex antioxidant defense systems (Ismail et al. 2016) (Fig. 8.1). Antioxidants can be classified into three broad divisions: water-soluble compounds (reductants, ascorbate); lipid-soluble compounds (α-tocopherol, β-carotene); and enzymes (superoxide dismutase [SOD], catalase [CAT], peroxidase [POD], ascorbate peroxidase [APX], and glutathione reductase [GR]) (Chen and Dickman 2005; Gill and Tuteja 2010).

Fig. 8.1
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Major plant antioxidants and their reactive oxygen species (ROS)–scavenging effects

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8.2.1 Enzymatic Antioxidants

Among the different antioxidant enzymes, SOD is the most effective intracellular enzymatic antioxidant that is ubiquitous in all aerobic organisms. SOD can catalyze and reduce O2 − to H2O2 because of its dismutation. SOD also indirectly reduces the risk of OH− formation by using O2 −. SOD catalyzes the first step of the enzymatic defense mechanism: the conversion of superoxide anions to hydrogen peroxide and water. If superoxide anions are not neutralized, oxidation occurs and hydroxyl radicals are formed. Hydroxyl radicals are extremely harmful because they are very reactive. Importantly, hydroxyl radicals cannot be scavenged by enzymatic means. Hydrogen peroxide can be decomposed by the activity of CATs and several classes of PODs, which act as important antioxidants.

CAT is another important enzymatic antioxidant; it is a tetrameric heme containing enzymes with the potential to directly dismutate H2O2 into H2O and O2, and it is indispensable for ROS detoxification during stressed conditions (Garg and Manchanda 2009). CAT has the highest turnover rate: one molecule of CAT can convert about 6 million molecules of H2O2 to H2O and O2 per minute. CAT enzymes remove H2O2 from peroxisomes by oxidases involved in β-oxidation of fatty acids, photorespiration, and purine catabolism. Different CAT isozymes have been reported in different plant species: two isozymes in barley (Azevedo et al. 1998), four in sunflower (Azpilicueta et al. 2007), and 12 in Brassica (Frugoli et al. 1996). Scandalias (1990) found three CAT isozymes in maize: CAT1 and CAT2 are localized in peroxisomes and the cytosol, whereas CAT3 is mitochondrial. It has also been reported that apart from reacting with H2O2, CAT also reacts with some hydroperoxides such as methyl hydrogen peroxide (MeOOH) (Ali and Alqurainy 2006). Overexpression of CAT encoded by the katE gene in rice conferred salt-induced oxidative stress tolerance (Nagamiya et al. 2007). Similarly, increased activity of CAT has been reported in chickpea following salt stress (Eyidogan and Oz 2005; Kukreja et al. 2005). Nonetheless, Srivastava et al. (2005) reported a decrease in CAT activity in Anabaena doliolum under NaCl and Cu2+ stress. Pan et al. (2006) studied the combined effect of salt and drought stress and found that it decreased CAT activity in Glycyrrhiza uralensis seedlings.

Thioredoxin and thiol-based glutathione are also important enzymes that play a crucial role in redox regulation. Thioredoxin includes a pleiotropic reduced NADP (NADPH)–dependent disulfide oxidoreductase, which catalyzes the reduction of exposed protein S–S bridges. Because of its dithiol-to-disulfide exchange activity, thioredoxin—acting as a hydrogen donor—determines the oxidation state of protein thiols (Lu and Holmgren 2014). This small 12 kDa protein contains a characteristic conserved catalytic Trp–Cys–Gly–Pro–Cys–Lys site. The two cysteine residues within this site can be oxidized reversibly to form a disulfide bridge. A specific selenoenzyme, thioredoxin reductase, is able to reduce the disulfide bond, utilizing NADPH as a hydrogen donor. The glutaredoxin system utilizes glutathione (a cysteine-containing tripeptide) in a manner similar to that used by the thioredoxin system. The antioxidant function of glutathione is implicated through two general mechanisms of reaction with ROS.

Glutathione peroxidase (GPX) belongs to a large family of diverse isozymes that use glutathione to reduce H2O2 and organic and lipid hydroperoxides, and therefore help plant cells with oxidative stress (Noctor et al. 2002). GPX uses glutathione to eliminate H2O2 and to decrease hydroperoxidation of lipids. Thus, these enzymes regulate intracellular levels of ROS such as H2O2 and O2 − (Gabbita et al. 2000). Millar et al. (2003) identified a family of seven related proteins—named AtGPX1–AtGPX7—in the cytosol, chloroplast, mitochondria, and endoplasmic reticulum of Arabidopsis. Upregulation of the GPX gene was noted in response to 1O2, showing the involvement of GPX in quenching 1O2 (Leisinger et al. 2001). It was noted that GPX activity in transgenic cotton seedlings was 30–60% higher under normal conditions but no different from GPX activity in wild-type seedlings under salt stress conditions (Light et al. 2005).

8.2.2 Nonenzymatic Antioxidants

Ascorbic acid (ASC) is the most abundant, powerful, and water-soluble antioxidant that acts to prevent or minimize the damage caused by ROS in plants (Smirnoff 2005; Athar et al. 2008). ASC is considered a most powerful ROS scavenger because of its ability to donate electrons in a number of enzymatic and nonenzymatic reactions. It can provide protection to membranes by directly scavenging O2 − and OH−, and by regenerating α-tocopherol from the tocopheroxyl radical. In chloroplasts, ASC acts as a cofactor of violaxantin de-epoxidase, thus sustaining dissipation of excess excitation energy (Smirnoff 2005). In addition to the importance of ASC in the ascorbate–glutathione cycle, it also plays an important role in preserving the activity of enzymes that contain prosthetic transition metal ions (Noctor and Foyer 1998). The ASC redox system consists of L-ascorbic acid (L-ASC), monodehydroascorbic acid (MDHA), and dehydroascorbic acid (DHA). Both oxidized forms of ASC are relatively unstable in aqueous environments, while DHA can be chemically reduced by glutathione.

Tripeptide glutathione (glu–cys–gly) is one of the crucial metabolites in plants and is considered a most important intracellular component of defense against ROS-induced oxidative damage. It occurs abundantly in the reduced form in plant tissues and is localized in all cell compartments, including the cytosol, endoplasmic reticulum, vacuole, mitochondria, chloroplasts, and peroxisomes, as well as in the apoplast (Mittler and Zilinskas 1992; Jiménez et al. 1998). Glutathione provides a substrate for multiple cellular reactions that yield GSSG (i.e., two glutathione molecules linked by a disulfide bond). The balance between glutathione and GSSG is a central component in maintaining the cellular redox state (Foyer and Noctor 2005). Glutathione is necessary to maintain the normal reduced state of cells so as to counteract the inhibitory effects of ROS-induced oxidative stress (Meyer 2008). It is a potent scavenger of 1O2, H2O2, and most dangerous ROS such as OH− (Op den Camp et al. 2003).

Proline is considered a potent antioxidant and a potential inhibitor of programmed cell death (Chen and Dickman 2005). Free proline has been proposed to also act as an osmoprotectant, a protein stabilizer, and a metal chelator, and helps in scavenging ROS (Ashraf and Foolad 2007; Trovato et al. 2008). In plants, there are two different precursors for proline. The first pathway is from glutamate, which is converted to proline by two successive reductions catalyzed by pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), respectively. P5CS is a bifunctional enzyme catalyzing first the activation of glutamate by phosphorylation and second the reduction of the labile intermediate c-glutamyl phosphate into glutamate semialdehyde, which is in equilibrium with the P5C form (Hu et al. 1992). An alternative precursor for proline biosynthesis is ornithine, which can be transaminated to P5C by ornithine-δ-aminotransferase (Orn-δ-OAT), a mitochondrially located enzyme. The glutamate pathway is the main pathway during osmotic stress. However, in young Arabidopsis plants, the ornithine pathway also seems to contribute and δ-OAT activity is enhanced (Roosens et al. 1998). Sorbitol, mannitol, myo-inositol, and proline have been tested for OH−-scavenging capacity, and it was found that proline appeared to be an effective scavenger of OH− (Smirnoff and Cumbes 1989; Gill and Tuteja 2010). Therefore, proline is not only an important molecule in redox signaling but also an effective quencher of ROS formed under salt stress (Alia and Saradhi 1991; Tanveer and Shah 2017). Chen and Dickman (2005) showed that addition of proline to DARas mutant cells effectively quenched ROS and prevented cell death by inhibiting ROS-mediated apoptosis. Enhanced synthesis of proline under drought or salt stress has been implicated as a mechanism in alleviation of cytoplasmic acidosis and maintenance of the NADPH:NADP+ ratio at a value compatible with metabolism (Hare and Cress 1997). Moreover, proline could maintain a low NADPH:NADP+ ratio, decrease 1O2 production from photosystem I (PSI), and lessen 1O2 and OH− damage to photosystem II (PSII) under stress (Szabados and Savouré 2010). Furthermore, proline has been reported to perform various antioxidant functions including (1) reductions in OH−, H2O2, and 1O2; (2) maintenance of a low NADPH:NADP+ ratio to decrease 1O2 production; (3) assistance in preventing programmed cell death; (4) reduction of detrimental effects of ROS on PSII; and (5) stabilization of mitochondrial respiration to protect the complex II phase of the electron transport chain in mitochondria (Szabados and Savouré 2010). Exogenously applied proline reduced the leaking of K+ by reducing the production of hydroxyl radicals in Arabidopsis roots (Cuin and Shabala 2007).

Glycine betaine accumulates predominantly in chloroplasts and protects the photosynthetic apparatus during oxidative stress (Ashraf and Foolad 2007). In addition, glycine betaine stabilizes the structure and function of the oxygen-evolving complex of PSII and protects the photosynthetic apparatus under high salt stress (Papageorgiou and Murata 1995). The introduction of genes synthesizing glycine betaine into nonaccumulators of glycine betaine has been shown to be effective in increasing tolerance of various abiotic stresses (Sakamoto and Murata 2002). In addition to the direct protective roles of glycine betaine, either through positive effects on enzyme and membrane integrity or as an osmoprotectant, glycine betaine may also protect cells from environmental stresses indirectly by participating in signal transduction pathways (Subbarao et al. 2000). Even in small concentrations, glycine betaine is very effective in stress amelioration. Exogenous application of glycine betaine (even in a submillimolar concentration) alleviates OH−-generated potassium ion leakage (Cuin and Shabala 2007), improving salinity stress tolerance. Consistent with this observation, halophytes (the most salt-tolerant species on the planet) may accumulate significant amounts (up to 90 μmol on a dry weight basis) of glycine betaine and thus can better withstand oxidative damage under saline conditions (Rhodes and Hanson 1993; Flowers and Colmer 2008). Transgenic crops overexpressing halophyte betaine aldehyde dehydrogenase (BADH)—a glycine betaine–synthesizing enzyme—were shown to possess enhanced salt and drought tolerance (Fitzgerald et al. 2009). Interestingly, overexpression of a plastid BADH in the salt-sensitive carrot resulted in remarkable salt tolerance (up to 400 mM NaCl), similar to that of halophytes (Kumar et al. 2004). The above findings could be attributed to both the osmotic role of glycine betaine and its ROS-scavenging ability.

Flavonoids are usually accumulated in the plant vacuole as glycosides, but they also occur as exudates on the surfaces of leaves and other aerial plant parts. Flavonoids can be classified into flavonols, flavones, isoflavones, and anthocyanins on the basis of their structure. Flavonoids are among the most bioactive plant secondary metabolites. Flavonoids serve as ROS scavengers by locating and neutralizing radicals before they damage the cell, which is important for plants to regulate redox potential under stressful conditions (Løvdal et al. 2010). Flavonoids function by virtue of the number and arrangement of their hydroxyl groups attached to ring structures. Their ability to act as antioxidants depends on the reduction potentials of their radicals and the accessibility of the radicals. Most flavonoids outperform well-known antioxidants, such as ASC and α-tocopherol (Hernandez et al. 2009).

Tocopherols are considered major antioxidants in biomembranes. The antioxidant ability of tocopherols against Fe2+ ascorbate–induced lipid peroxidation declined in the order of α > β ≈ γ > δ, with each single molecule of these tocopherols protecting up to 220, 120, 100, and 30 molecules of polyunsaturated fatty acids, respectively, before being consumed (Fukuzawa et al. 1982). Tocopherols have been shown to prevent the chain propagation step in lipid auto-oxidation, which makes them an effective free-radical trap. α-Tocopherol (vitamin E) detoxifies 1O2 and lipid peroxyl radicals, thus preventing lipid peroxidation under abiotic stress (Szarka et al. 2012). Among the different isoforms of tocopherols, α-tocopherol is the predominant form in plant green tissues. This isoform is synthesized in a plastid envelope and is stored in plastoglobuli of the chloroplast stroma and in thylakoid membranes, suggesting that α-tocopherol is pivotal to decreased production of ROS (primarily that of 1O2) in the chloroplast during environmental stresses (Szarka et al. 2012). Recently, it has been found that oxidative stress activates the expression of genes responsible for the synthesis of tocopherols in higher plants (Wu et al. 2007). Increased levels of α-tocopherol and ASC have been found in tomato and in A. doliolum, helping to protect membranes from oxidative damage (Hsu and Kao 2007).

α-Tocopherol can scavenge 1O2 in two ways. First, α-tocopherol quenches 1O2 physically via resonance energy transfer (Fahrenholtz et al. 1974); following this, 1O2 scavenging can happen through a chemical reaction (Falk and Munné-Bosch 2010). During the resonance energy transfer mechanism of 1O2 scavenging, α-tocopherol content is not altered significantly and one molecule of α-tocopherol can deactivate 120 molecules of 1O2, whereas the chemical scavenging mechanism implies involvement of an intermediate hydroperoxydienone that decomposes to form tocopherol quinone and tocopherol quinone epoxides, resulting in a significant decline in the α-tocopherol content (Munne-Bosch and Alegre 2002). A comparison between a halophyte (Cakile maritima) and a glycophyte (Arabidopsis thaliana) showed that C. maritima may detoxify salt-induced 1O2 production through direct quenching, because the α-tocopherol level did not change significantly during salt stress. At the same time, A. thaliana achieved the same result through a chemical reaction, showing a reduction of 50% in the production of α-tocopherol (Ellouzi et al. 2011). α-Tocopherols also function as recyclable chain reaction terminators of polyunsaturated fatty acid (PUFA) radicals generated by lipid oxidation (Hare et al. 1998). α-Tocopherols scavenge lipid peroxy radicals and yield a tocopheroxyl radical that can be recycled back into the corresponding α-tocopherol by reacting with ascorbate or other antioxidants (Igamberdiev and Hill 2004).

8.3 Targeting Redox Regulation and Antioxidant Defense Systems in Breeding Programs

The number of papers linking oxidative stress with salinity has increased exponentially over the past two decades. There have also been reports of increased antioxidant activity in plants grown under saline conditions (Hernandez et al. 2000; Sairam and Srivastava 2002). It is hardly surprising that the idea of improving salinity stress tolerance by increased antioxidant production is gaining momentum (Table 8.1). However, many other reports have questioned the validity of this approach, reporting either no correlation or a negative correlation between the activity of antioxidant enzymes and plant salinity stress tolerance (Tables 8.2 and 8.3). The possible reasons for this discrepancy most likely lie in the fact that some ROS such as H2O2 play a very important signaling role in adaptive and developmental responses, and so tampering with them may result in pleiotropic effects (De Pinto et al. 2006). It is becoming increasingly evident that considerable variations exist in the production of both enzymatic and nonenzymatic antioxidants in response to salt stress in various plant tissues and at various time points. Hence, the interspecific or intraspecific aspects of ROS production and scavenging should be taken into account. Last, but not least, the diversity of known antioxidants should be accounted for. Some supporting arguments are given below.

Table 8.1 Selected examples of reported positive correlations between expression of antioxidant genes and salt stress tolerance in transgenic plant species
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Table 8.2 Stress-induced changes in antioxidant activity of major enzymatic antioxidants in different plant species
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Table 8.3 Stress-induced changes in antioxidant activity of major nonenzymatic antioxidants in different plant species
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8.3.1 Antioxidant Activity at the Tissue/Organ Level

Activation of the antioxidant defense system occurs via enzymatic and nonenzymatic mechanisms against salt stress. The antioxidant responses, though, are different in different organs and/or tissues (Turan and Tripathy 2013). Hamada et al. (2016) found that the total antioxidant capacity and polyphenol content in maize increased with increased salinity levels in roots and mature leaves but showed no changes in young leaves. They also showed that SOD, APX, glutathione (GSH), glutathione S-transferase (GST), and ASC content increased particularly in maize roots, while total tocopherol levels increased specifically in shoot tissues. Proline content was slightly decreased in young leaves in maize plants but did not show significant changes in maize roots and mature leaves under exposure to salinity stress (Hamada et al. 2016). Other studies have also reported tissue-specific responses of some other antioxidants. For example, ASC and tocopherol content has been shown to increase in the leaves of tomato to protect them against oxidative stress under high salinity (Salama et al. 1994; Tuna 2014); however, the levels of ASC and tocopherols declined in rice leaves under salinity stress (Turan and Tripathy 2013). There was no significant change in proline content in Sorghum bicolor leaves under salinity stress, but proline content in Sorghum sudanense decreased slightly with salinity (De Oliveira et al. 2013). Redox changes estimated by the ratios of redox couples (ASC:total ascorbate and GSH:total glutathione) showed significant decreases in maize roots (Hamada et al. 2016). Tocopherol, on the other hand, appears to be a more shoot-specific antioxidant in maize seedlings (Hamada et al. 2016). In lentil, root tissues were less affected by salt stress and had higher activity of Cu/ZnSOD, APX, and GR in roots than in shoots (Bandeoğlu et al. 2004). In common bean (Phaseolus vulgaris), salinity stress led to reductions in the activity of SOD and APX in nodules but not in the bulk of the roots (Jebara et al. 2005). These conflicting results could be due to plant/tissue specificity. Moreover, plant age–related differences in antioxidant responses in plant cells, tissues, and organs could also explain the reason behind the interspecific or intraspecific aspects of ROS production and antioxidant activity. For instance, mature maize leaf cells (in the distal leaf parts) are more sensitive to high salinity than younger cells (from actively expanding leaf parts), which have higher antioxidant activity (Kravchik and Bernstein 2013). These studies have indicated possible roles of ROS in the systemic signaling from roots to leaves and activation of antioxidants for better protection against oxidative stress and/or salt stress.

8.3.2 Antioxidant Activity at the Organelle Level

Besides tissue-specific and/or organ-specific responses, organelle-specific responses of antioxidants under salt stress have been observed. The activity of different antioxidants within different compartments of a cell, tissue, or organ plays a role in protection against oxidative stress caused by salt stress. Hernandez et al. (2000) showed that salt stress tolerance in pea was associated with high SOD activity in the apoplast and high activity of dehydroascorbate reductase (DHAR), GR, and monodehydroascorbate reductase (MDHAR) in the symplast. Moreover, Mittova et al. (2004) showed that the mitochondria and peroxisomes of salt-treated roots of wild tomato had increased levels of lipid peroxidation and H2O2, coupled with decreased activity of SOD, POD, ASC, and GSH, suggesting that improved endogenous production of antioxidants in the mitochondria and/or peroxisome could improve salt stress tolerance. In another study, higher activity of SOD, APX, and GR was noted in the chloroplastic fraction as compared with the mitochondrial fraction and cytosolic fraction (Sairam and Srivastava 2002).

8.3.3 Antioxidant Activity at Different Time Points

Antioxidant activity also shows a pronounced time dependence and thus may be different at various time points. Mhadhbi et al. (2011) reported increased activity of CAT, SOD, and POD in salt-treated Medicago truncatula roots at 24 h; however, this activity was lost after 48 h of salt stress. In pea, SOD activity increased after 48 h of salt stress, while GR showed an increase after 24 h of salt stress Hernandez et al. (2000). These authors also found no change in APX during salt stress at any time point. Shalata et al. (2001) showed that under long-term salt stress conditions, the activity of SOD, APX, CAT, and MDHAR was increased and reached a maximum 16 days after the beginning of salinization, and then decreased, while GR activity decreased from the start of salt stress. All of these studies reported highly varied responses and production of antioxidants at various time points under salt stress, and suggested that the variations could have been due to differences in experimental conditions, data collection, and plant species.

8.3.4 Targeting Quantitative Trait Loci for Antioxidant Activity

The practical aspect of targeting antioxidant activity in breeding programs should also be considered. Jiang et al. (2013) conducted quantitative trait locus (QTL) analysis of the activity of antioxidant enzymes and malondialdehyde content in wheat seeds during germination. These authors discovered 22 unconditional QTLs on 1A, 1B, 1D, 2B, 2D, 3A, 4A, 6B, 7A, and 7B, scattered on nine chromosomes. Eight of these QTLs were for SOD activity, five for POD, and six for CAT. In rice, two QTLs for malondialdehyde (MDA) content in rice leaves were detected on chromosome 1, with additive effects from maternal and paternal parents accounting for 4.33% and 4.62% of phenotype variations, respectively. Rousseaux et al. (2005) found 20 QTLs in tomato; of these, five were for total antioxidant activity, five for ASC, and nine for total phenolics. Frary et al. (2010) carried out QTL analysis and found nine QTLs for phenolic compounds and 14 for flavonoids in tomato. Given these numbers, it will be very difficult—if it is at all possible—to make a valid recommendation to breeders as to which of these QTLs play bigger role(s) and thus should be transferred into high-yielding varieties to increase their stress tolerance.

It has also been argued (Bose et al. 2014) that truly salt-tolerant plants do not allow formation of harmful ROS in the first instance and, as such, require no higher antioxidant activity. This can be achieved by increasing the rate of sodium exclusion from the cytosol, either into vacuoles (via mechanisms including NHX tonoplast Na+/H+ exchange) (Blumwald 2000) or into the apoplast (via SOS1-mediated Na+ exclusion from the cell) (Shi et al. 2000). Halophytes show high levels of salt stress tolerance and possess high levels of antioxidant production at an intrinsic level. As described in Sects. 8.1 and 8.2.1, SOD rapidly converts O2 – to H2O2 at the initial level of salt stress and the latter plays an important role as a second messenger, triggering cascades of different adaptive responses at the genetic and physiological levels; thus, rapid conversion of O2 – to H2O2 could be essential for early defense in halophytes. Nonetheless, it still remains to be established how the stress-induced increase in H2O2 production is ultimately converted into plant adaptive responses (Miller et al. 2010). Because of similarities between Ca2+- and H2O2-induced signatures, the roles of other enzymatic antioxidants may be attributed to the need to decrease the basal levels of H2O2 once the signaling has been processed. In this context, the roles of CAT and APX in shaping the H2O2 signature may be similar to those in Ca2+ efflux systems (Bose et al. 2011).

8.4 Concluding Remarks

ROS are produced as a result of a large number of metabolic processes, occurring at both intracellular and extracellular locations. Plants possess different enzymatic and nonenzymatic antioxidants to scavenge ROS, regarded as redox regulatory mechanisms in plants. Different antioxidants have different ROS-scavenging effects (Fig. 8.1), and their effects are highly tissue specific and organelle specific. A significant variability exists among different plant species and/or among different genotypes of the same plant species in terms of the kinetics of antioxidant production and activity. Different reports have described controversial results regarding increases or decreases in the activity of different antioxidants in response to salinity. In the light of this, it appears not to be highly fruitful to try and improve plant stress tolerance by increasing the activity of some specific antioxidants via either genetic engineering or a MAS-based approach, without accounting for the tissue and time dependence of this process. There is a need for better understanding of the role of ROS as signaling components of plant adaptive mechanisms, and development of tools for targeted regulation of plant redox systems in specific cellular compartments and tissues.