**4. Salt tolerance approaches**

Considering the adverse effects of salt stress in wheat plant, biologists are trying to find out the salt-tolerant strategies in plants by different approaches. Many researchers found positive effect in using exogenous phytoprotectants in alleviating salt-induced damages in wheat. In this section, some of the evidences are discussed.

#### **4.1. Use of osmoprotectants**

To prevent the adverse effects of various environmental stresses including salt stress, plants demonstrate a variety of adaptive mechanisms both at the cellular and organismal levels. Under salt stress conditions, to cope with the salt-induced osmotic, ionic as well as oxidative stresses, plant synthesizes and accumulates organic compatible solutes or osmolytes [48, 65, 66]. Accumulation of these compatible solutes is one of the most important physiological strategies employed by plants under salt stress conditions. Osmoprotectants or osmolytes are small, highly soluble, uncharged, and nontoxic organic molecules which help to survive organisms in extreme osmotic stresses. Osmoprotectants comprise of (i) α-amino acids such as proline (Pro) and ectoine; (ii) ammonium compounds such as glycine betaine (GB), β-alanine betaine, dimethylsulfoniopropionate (DMSP), and choline; and (iii) polyols, sugars, and sugar alcohols such as trehalose (Tre), sorbitol, and mannitol, etc. These osmoprotectants perform vital functions in osmotic adjustment, stabilizing proteins and membranes. Thus, enhanced salt stress tolerance is observed in plants overexpressing the osmoprotectants biosynthetic and metabolic genes. Enhanced salt exposure causes increased biosynthesis of osmoprotectants (Pro, GB, Tre, ecotine, and sorbitol, etc.) which provides enhanced osmotic stress tolerance generated from salt stresses [67, 68] (**Table 3**). For mitigating salt-induced damages, in recent decades, the use of exogenous osmoprotectants has been found effective [12, 69]. Several research findings demonstrated that the use of osmoprotectants provided significant protection against adverse effects of salt stresses in *T. aestivum* seedlings (**Table 3**). At the same time, several research studies proved Pro as a potent protectant against the adverse effects of salt. Proline acts not only in osmotic adjustment as a compatible solute, but also in scavenging ROS, chelating metal, activating detoxification pathways, balancing cells redox status, buffering cytosolic pH, storing energy (carbon and nitrogen), stabilizing subcellular membranes and structures including photosystem II (PS II), and as signaling molecule [70–74]. Raza et al. [75] demonstrated the effect of exogenous GB (50 mM and 100 mM) in moderately salt-sensitive (MH-97) and salt-tolerant (S-24) wheat cultivars grown under salt stress (15 dS m−1 NaCl). Glycine betaine treatment ameliorated the salt-induced photosynthetic reduction as well as increased the photosynthetic capacity, water use efficiency, and osmotic adjustment where salt-tolerant (S-24) cultivar showed better performance against salt stress compared to moderately salt-sensitive (MH-97) cultivar. Later, with the same experimental procedure, they again suggested that the exogenous GB modulated the activities of antioxidant enzymes such as SOD, CAT, and POD which contributed significantly to salt stress tolerance in *T. aestivum* [76]. It has been reported that accumulation of Pro protects *T. aestivum* from the salt-induced damages by maintaining a higher K+ /Na+ ratio and reducing ionic toxicity [38], increasing the major antioxidant enzymes (CAT, APX, SOD, and POD) activities [77]. In *T. aestivum*, GB (10 mM and 30 mM) supplementation with salt stress (150 mM NaCl) increased the germination percentage, shoot Ca content, total chl content, and thus confer salt stress tolerance [78]. Khan et al. [79] reported that increased grain yield in *T. aestivum* associated with the increased Pro, chl content and K+ /Na+ ratio. Overexpression of GB in transgenic *T. aestivum* lines T1, T4, T6, and Shi 4185 (wild type line) caused enhanced salt stress (200 mM NaCl) tolerance by enhancing ROS scavenging, osmotic adjustment and regulating ion homeostasis [80]. Salt stresses (10 dS m−1 NaCl) were imposed in two wheat cultivars (cv. Seher and Lasani). In both wheat cultivars, salt stresses caused significant reduction in the germination percentage, chl contents and growth. Exogenous Pro (50 and 100 mM) application alleviated the adverse effects of salt stress by improving the germination percentage, seedling growth and chl contents of wheat plants but 100 mM Pro was found more effective compared to 50 mM Pro [81]. Mahboob et al. [82] reported that the supplementation of Pro (50 and 100 mM) ameliorated the salt (60 and 120 mM NaCl) induced reduction of plant growth, photosynthetic pigments and ionic balance by increasing shoot and root length, chl *a*, *b* contents*,* FW and DW of seedlings and endogenous Pro, GB, and K+ /Na+ ratio in *T. aestivum* seedlings. Exogenous Pro (60 ppm) upregulated the endogenous hormones (gibberelic acid (GA3 ) and indole acetic acid (IAA)), ammonium compounds (GB and choline) and downregulated the MDA content and growth inhibitor abscisic acid (ABA) in salt stressed *T. aestivum* [83]. Salt (50, 150, and 300 mM) induced disruption of photosynthetic pigments and protein polypeptide synthesis in *T. aestivum* were prevented by the exogenously applied Pro (50 ppm) and at the same time by protecting the turnover machinery of proteins [84]. Besides osmotic adjustment, GB is also involved in ROS scavenging, stabilizing macromolecules (nucleic acids, proteins, and lipids) and various components of photosynthetic machinery such as PS II complexes and RuBisCO and acts as reservoir of carbon and nitrogen sources [85–87]. Upon salt exposure (150 mM NaCl), reduced lipid peroxidation, increased glutathione (GSH) and GB concentrations, enhanced plasma membrane protection, increased cell solute potential and improved ion homeostasis were observed when caryopsis of *T. aestivum* were primed with GB (25, 50, 100 mM) [88]. Increasing the K+ /Na+ and Ca+ /Na+ ratios, reducing MDA content, protecting photosynthetic apparatus, improving plasma membrane integrity and stabilizing macromolecules (proteins, PS II and transporters) GB (20 mM) imparted in salt stress tolerance in *T. aestivum* [83]. Exogenous GB (5 mM) application improved chl *a*, total chl and K+ content of roots, increased root length, plant height, FW and DW of *T. aestivum* under salt stresses (100 and 200 mM NaCl) [89]. Rao et al. [57] suggested that the enhanced production of Pro and GB in six salt-tolerant cultivars (*T. aestivum* cv. AUQAB-2000, PUNJAB-85, PIRSABAK-05, BAKHAR-2002, FARKHARE-SARHAD and KAGHAN-94) alleviated the damaging effects of salt stress by activating their antioxidant enzymes. Endogenous Pro and GB mediated salt stress (8 EC, 16 EC) mitigation in fifteen *T. aestivum* cultivars were further reported by Rao et al. [57]. They suggested that the five cultivars of wheat (SEHAR-2006, LU26-CTR, NARC-2009, BARS-2009, PIRSABAK-09) showed obvious salt stress tolerance by increasing the production of Pro and GB. Yan and Zheng [90] demonstrated that pretreatment with Tre (2, 20, and 40 mM) alleviated the adverse effects of salt stress (3 g L−1 NaCl) in *T. aestivum* cv. Yangmai-19. Various beneficial effects were observed in different physiological parameters. Increased relative growth rate, relative chl content, N content, DW and biomass plant-1 were observed with Trehalose supplementation. Trehalose application also improved Pro accumulation, K+ accumulation and K+ /Na+ ratio. In addition,

**4. Salt tolerance approaches**

160 Wheat Improvement, Management and Utilization

**4.1. Use of osmoprotectants**

ages by maintaining a higher K+

/Na+

antioxidant enzymes (CAT, APX, SOD, and POD) activities [77]. In *T. aestivum*, GB (10 mM and

ratio and reducing ionic toxicity [38], increasing the major

this section, some of the evidences are discussed.

Considering the adverse effects of salt stress in wheat plant, biologists are trying to find out the salt-tolerant strategies in plants by different approaches. Many researchers found positive effect in using exogenous phytoprotectants in alleviating salt-induced damages in wheat. In

To prevent the adverse effects of various environmental stresses including salt stress, plants demonstrate a variety of adaptive mechanisms both at the cellular and organismal levels. Under salt stress conditions, to cope with the salt-induced osmotic, ionic as well as oxidative stresses, plant synthesizes and accumulates organic compatible solutes or osmolytes [48, 65, 66]. Accumulation of these compatible solutes is one of the most important physiological strategies employed by plants under salt stress conditions. Osmoprotectants or osmolytes are small, highly soluble, uncharged, and nontoxic organic molecules which help to survive organisms in extreme osmotic stresses. Osmoprotectants comprise of (i) α-amino acids such as proline (Pro) and ectoine; (ii) ammonium compounds such as glycine betaine (GB), β-alanine betaine, dimethylsulfoniopropionate (DMSP), and choline; and (iii) polyols, sugars, and sugar alcohols such as trehalose (Tre), sorbitol, and mannitol, etc. These osmoprotectants perform vital functions in osmotic adjustment, stabilizing proteins and membranes. Thus, enhanced salt stress tolerance is observed in plants overexpressing the osmoprotectants biosynthetic and metabolic genes. Enhanced salt exposure causes increased biosynthesis of osmoprotectants (Pro, GB, Tre, ecotine, and sorbitol, etc.) which provides enhanced osmotic stress tolerance generated from salt stresses [67, 68] (**Table 3**). For mitigating salt-induced damages, in recent decades, the use of exogenous osmoprotectants has been found effective [12, 69]. Several research findings demonstrated that the use of osmoprotectants provided significant protection against adverse effects of salt stresses in *T. aestivum* seedlings (**Table 3**). At the same time, several research studies proved Pro as a potent protectant against the adverse effects of salt. Proline acts not only in osmotic adjustment as a compatible solute, but also in scavenging ROS, chelating metal, activating detoxification pathways, balancing cells redox status, buffering cytosolic pH, storing energy (carbon and nitrogen), stabilizing subcellular membranes and structures including photosystem II (PS II), and as signaling molecule [70–74]. Raza et al. [75] demonstrated the effect of exogenous GB (50 mM and 100 mM) in moderately salt-sensitive (MH-97) and salt-tolerant (S-24) wheat cultivars grown under salt stress (15 dS m−1 NaCl). Glycine betaine treatment ameliorated the salt-induced photosynthetic reduction as well as increased the photosynthetic capacity, water use efficiency, and osmotic adjustment where salt-tolerant (S-24) cultivar showed better performance against salt stress compared to moderately salt-sensitive (MH-97) cultivar. Later, with the same experimental procedure, they again suggested that the exogenous GB modulated the activities of antioxidant enzymes such as SOD, CAT, and POD which contributed significantly to salt stress tolerance in *T. aestivum* [76]. It has been reported that accumulation of Pro protects *T. aestivum* from the salt-induced damTre has functions in stabilizing the biomolecules and structures like membrane lipids, proteins under salt stress [91–93]. Salt-sensitive wheat cultivar (*T. aestivum* cv. Kızıltan-91) under salt stress (100 mM NaCl) showed physiological alteration. However, pretreatment with exogenous mannitol (100 mM) reversed the deleterious salt effects by increasing antioxidant enzymes (such as SOD, POD, CAT, APX, and GR) activities, appearance of SOD and POD isozyme activity bands and reducing lipid peroxidation [59].


romolecules (proteins, PS II, and transporters)


**Table 3.** Protective effects of various exogenously applied osmoprotectants under salt stress in *T. aestivum*.

Priming of *T. aestivum* seeds with choline (5 and 10 mM) reduced the damaging effects of NaCl (150 mM) by increasing the K+ , Ca2+, GB accumulation, improved ion homeostasis and decreased Na+ and Cl<sup>−</sup> in both shoot and root, mitigated PM permeability and reduced lipid peroxidation of leaf [94]. Expression of *mtlD* gene encoding mannitol-1-phosphate dehydrogenase resulted in enhanced salt stress tolerance in *T. aestivum* due to defensive roles of mannitol against salt stress [95]. The *mtlD* gene encoding mannitol-1-phosphate dehydrogenase transformation in *T. aestivum* cv. Giza 163 conferred salt stress tolerance by inducing mannitol and reducing sugars in tissues of plant [96]. Kerepesi et al. [97] demonstrated that increased fructan contents in salt resistant (Sa) and moderately salt-tolerant (Ch) varieties of *T. aestivum* showed increased tolerance against salt stress (200 mM NaCl). Sharbatkhari et al. [98] investigated the role of fructan in salt-tolerant (Bam) and salt-sensitive (Ghods) cultivars of *T. aestivum*. They found higher fructan accumulation and remobilization in salt-tolerant Bam cultivar, which contributed to the higher salt stress tolerance by increasing the photosynthetic capacity and decreasing the salt induced severe yield loss.

#### **4.2. Plant hormone**

Tre has functions in stabilizing the biomolecules and structures like membrane lipids, proteins under salt stress [91–93]. Salt-sensitive wheat cultivar (*T. aestivum* cv. Kızıltan-91) under salt stress (100 mM NaCl) showed physiological alteration. However, pretreatment with exogenous mannitol (100 mM) reversed the deleterious salt effects by increasing antioxidant enzymes (such as SOD, POD, CAT, APX, and GR) activities, appearance of SOD and POD

> • Increased shoot and root length • Increased FW and DW of seedlings

• Increased chl *a*, *b* contents • Improved Pro, GB, K+

substomatal CO2

CO2

tion rate)

• Improved WUE

and GR

damage

**Protective effects References**

contents, and K+

concentration, and transpira-

• Improved gaseous exchange parameters (net

• Increased chl *a*, *b*, and total chl contents

• Increase activities of SOD, POD, CAT, APX,

• Reduced lipid peroxidation and membrane

• Increased the GSH and GB concentrations • Enhanced plasma membrane (PM) protection

• Increased endogenous hormones (GA and IAA)

and Ca+

• Improved PM integrity and stabilization of macromolecules (proteins, PS II, and transporters)

/Na+ ratios

• Increased photosynthetic capacity • Increased stomatal conductance

• Reduced lipid peroxidation

• Increased the cell solute potential • Improved ion homeostasis

• Increased chl *a* and *b*

• Increased K+

• Increased GB and choline

• Reduced MDA content

• Decreased MDA content and ABA

/Na+

• Improved K, Ca, and Zn contents

• Protected photosynthetic apparatus

assimilation rate, stomatal conductance,

/Na+ ratio Mahboob et al. [82]

Talat et al. [81]

Raza et al. [75]

Seckin et al. [59]

Salama et al. [88]

Hendawey et al. [83]

Hendawey et al. [83]

isozyme activity bands and reducing lipid peroxidation [59].

**Doses of osmolytes**

50 and 100 mM Pro, foliar spray

50 mM and 100 mM Pro, foliar spray

50 mM and 100 mM GB, foliar spray

100 mM mannitol, pretreatment, 24 h

25, 50, and 100 mM GB, caryopsis priming, 24 h

60 ppm Pro, foliar spray

20 mM GB, foliar spray

**Cultivars Salinity doses** 

ESW-9525 and kherman

Seher and Lasani

MH-97 and S-24

Kızıltan-91 100 mM

Gomeza 7 150 mM

Sakha 93 and Gimmeza7

Sakha 93 and Gimmeza7

**and duration**

162 Wheat Improvement, Management and Utilization

60 and 120 mM NaCl, 7 d

10 dS m−1 NaCl, 6 d

15 dS m−1 NaCl

NaCl, 5 d

NaCl, 38 d

10.04 dS m−1 (soil), 35–65 d 7.33 dS m−1 (irrigation water), 35–65 d

10.04 dS m−1 (soil) 7.33 dS m−1 (irrigation water), 35 to 65 d

Plant hormones are chemicals produced within the plants at low concentration involved in regulation of plant development and tolerance towards various stresses including salinity [99]. Now-a-days, various kinds of plant hormones such as ABA, auxin, cytokinins (CK),




**Table 4.** Protective effects of various exogenously applied phytohormones under salt stress in *T. aestivum*.

#### **4.3. Plant nutrient**

brassinosteroids and GA3

164 Wheat Improvement, Management and Utilization

**Cultivars Salinity dose and duration**

(15 ds m−1)

150 mM NaCl, entire growth period

150 mM NaCl, entire growth period

50, 100, 150, and 200 mM NaCl),entire

15 dS m−1, entire life

life cycle

cycle

Auxin (Tryptopan) 4.89 × 10−4 mM, 12 h seed priming

4.89 × 10−1 mM auxin (tryptophan), 12 h seed

priming

spray

15 dS m−1, 8 d 100, 150 and 200 mg L−1

150 mg L−1 GA3

cytokinins (kinetin and BAP), 12 h seed priming

100, 150 and 200 mg L–1 cytokinins (kinetin and BAP), 12 h seed priming

, foliar

MH-97 (salt intolerant), Inqlab-91 (salt tolerant)

MH-97 (salt intolerant), Inqlab-91 (salt tolerant)

Sohag 3 (sensitive), Giza 168 (tolerant)

MH-97, Inqlab-91

MH-97, Inqlab-91

GA3

GA3

are externally used for alleviating various kinds of abiotic stresses

**Dose of phytohormones Protective effects References**

• Increased net assimilation rate

• Increased productive tiller and

assimilation rate

Iqbal and Ashraf [44]

Iqbal and Ashraf [100]

Shaddad et al. [102]

Iqbal et al. [105]

Iqbal and Ashraf [104]

• Increased CO2

• Increased growth

• Increased germination percentage

• Improved ion homeostasis • Increased shoot DW

• Improved leaf area, photosynthetic pigment, carobohydrate, protein, amino acid and Pro content, grain weight

• Increased germination rate • Increased early seedlings growth such as shoot DW and

• Increased plant height, shoot

1000-grain weight, grain yield

root DW

dry biomass • Increased fertile tiller,

grain yield

including salinity (**Table 4**). The plant growth hormone auxin increased the germination percentage, shoots DW and maintained ion homeostasis under salt stress condition [100]. Iqbal and Ashraf [101] reported that seed priming with different auxins alleviated salt stress (15 dS m–1) by maintaining hormonal balance and assimilation rate and improved growth and yield of both tolerant and sensitive cultivars under salt stress condition. Seed priming with

 alleviates the drastic effect of salinity and increases grain weight and grain quality by improving photosynthetic pigments, leaf area and plant growth [102]. Foliar application of

 also confers salt stress tolerance by increasing germination percentage, plant growth and upregulating antioxidant enzyme [103]. Seed priming with cytokinin such as kinetin and benzylaminopurine (BAP) increase germination percentage and grain yield by increasing plant growth, productive tiller and 1000-grain weight under salt stress condition [104, 105]. Gurmani et al. [106] noted that, seed priming with ABA improved salt stress tolerance by increasing net assimilation rate, chl content and decreasing Na uptake. It is also evident that phytohormone brassinosteroid plays role in alleviating salt stress. Ali et al. [107] reported that brassinosteroid increased grain yield by improving photosynthetic attribute, assimilation rate and transpiration rate under salt stress condition (150 mM NaCl). Eleiwa et al. [22] also showed brassinosteroid-induced positive response in wheat seedlings under salt stress conditions (**Table 4**).

> Along with other physiological and biochemical functions, plant nutrients play positive roles in alleviating damage effects of abiotic stresses including salinity (**Table 5**). Exogenous application of K enhanced salt stress tolerance in wheat seedlings by improving photosynthetic pigments, antioxidant enzyme activity, K uptake and decreasing Na uptake [109, 110]. Foliar application of phosphorus (P) also alleviated salt-induced damage by increasing plant biomass, leaf area and decreasing Na uptake [111]. Application of CaSO4 increased plant growth, water status and K and Ca uptake under salt stress condition [112]. Later on, Tian et al. [113] noted that application of Ca(NO3 )2 reduced saltinduced oxidative damage by decreasing lipid peroxidation and electrolyte leakage in wheat seedlings.


**Table 5.** Protective effects of plant nutrients under salt stress in *T. aestivum*.

#### **4.4. Antioxidant**

Antioxidants are important for plants to maintain the ROS level lower. Plant possesses various non-enzymatic antioxidants in their cellular components to protect themselves from oxidative stress. The major antioxidant includes AsA, GSH, tocopherol and some phenolic compounds. Some of these antioxidants showed advanced protection against salt-induced oxidative stress when they were applied exogenously (**Table 6**). However, these are mostly dose dependent. A number of studies have been reported the positive effects of AsA in mitigating salt stress in wheat. Athar et al. [20] studied the effect of AsA on wheat plants subjected to salt stress. Salt stress (150 mM NaCl) caused reduction in growth and photosynthesis which were associated with decrease in tissue K+ /Na+ ratio in both sensitive and moderately tolerant varieties. However, root applied AsA (100 mg L−1) counteracted the adverse effects of salt stress on the growth of tolerant variety which was due to the enhanced endogenous AsA level and CAT activity, and higher photosynthetic capacity, and accumulation of K+ and Ca2+ in the leaves. Their study supports the notion that exogenous AsA counteracts the adverse effects of salt stress on growth of wheat by improving photosynthetic capacity of wheat plants against salt-induced oxidative stress and maintaining ion homeostasis, however, these effects were cultivar specific [20]. Ascobin (compound composed of ascorbic acid and citric acid) was found to be effective in mitigating salt-induced damages in wheat as reported by Elhamid et al. [114]. Salt stress markedly increased the lipid peroxidation while the activities of antioxidant enzymes (SOD, CAT, POD, APX and GR) dramatically increased. However, foliar treatment of wheat cultivars with ascobin could partially alleviate the harmful effect of salinity especially at the lower levels of salinity imposed in the two cultivars of wheat at most of the studied parameters [114]. Apart from the dose, mode of application is also a factor to initiate the protective effect by exogenous AsA. In their study Athar et al. [115] found differential effects when AsA was applied through the rooting medium, or as seed soaking or as foliar spray to salt stressed (120 mM NaCl) wheat plants. Exogenous AsA mitigated the adverse effect, e.g. improved leaf ascorbic acid, activities of CAT, POD, and SOD. Root applied AsA caused more enhancements in photosynthetic capacity and more reduction in leaf sodium (Na+ ) compared with AsA applied as seed soaking or foliar spray. However, the effects were also cultivar specific [115]. In a hydroponic experiment Khan et al. [116] showed that foliar applied AsA (50 and 100 mg L−1) could not alleviate the adverse effects of salt stress on plants, but it improved the growth of nonstressed plants. Since AsA failed to enhance the antioxidant defense, it enhanced the Na+ accumulation in the leaves but did not change the K+ accumulation in the salt-stressed plants. Azzedine et al. [21] observed that the exogenous AsA improved the plant growth under salt stress condition which was partly due to the increased leaf area, improved chl and carotenoid contents, enhanced Pro accumulation, and decreased H2 O2 content. Melatonin (*N*-acetyl-5-methoxytryptamine) is also considered a potential antioxidant in plants which is distributed in many parts of the plant. Due to its universal hydrophilic and hydrophobic nature and solubility in both water and lipid, it can cross cell membranes easily and enter subcellular compartments and hence, considered as an antioxidant and a modulator in multiple plant developmental processes and various stress responses [117]. In their pot experiment, Sadak et al. [117] observed that wheat seeds presoaked with melatonin (100 and 500 μM) provided better growth, photosynthetic pigments, yield, and quality in wheat under salinity (3.85 and 7.69 dS m−1). Melatonin treatments at different levels caused significant increase in yield and yield attributes, carbohydrate, protein, N, P, K, flavonoids, phenolic contents, and antioxidant activity either in nonstressed and salinity-stressed plants relative to their corresponding controls. Importantly, 500 μM melatonin was more effective than 100 μM. Farouk [118] reported that both AsA and α-tocopherol minimized salt-induced senescence of flag leaves of wheat. This was due to enhanced activities of antioxidant enzymes which led to the lower lipid peroxidation and H2 O2 accumulation. Exogenous antioxidants also decreased membrane permeability, Na and Cl content. These higher levels of antioxidants and lower level of H2 O2 in flag leaf might be the prerequisite for delayed leaf senescence in antioxidants-sprayed plants [118].

**4.4. Antioxidant**

**Cultivars Salinity dose and duration**

> 150 mM NaCl, 113 d

166 Wheat Improvement, Management and Utilization

40, 80, and 120 mM NaCl, 90 d

PUNJAB-85 50 mM NaCl, 34 d 3 and 6 mM

Jimai 22 100 mM NaCl, 15 d 17.5 mM

50, 100, 150 and 200 mM K2

25 and 150 mg

O kg–1 soil,

P L–1, foliar application

CaSO4

Ca(NO3

**Table 5.** Protective effects of plant nutrients under salt stress in *T. aestivum*.

)2, 15 d

106 d

K2

150 mM NaCl 400 and 800 mg

110 d

SO4 ,

NAYAB-11 and MILLAT-11

Gemiza 9, Sakha 93

Antioxidants are important for plants to maintain the ROS level lower. Plant possesses various non-enzymatic antioxidants in their cellular components to protect themselves from oxidative stress. The major antioxidant includes AsA, GSH, tocopherol and some phenolic compounds. Some of these antioxidants showed advanced protection against salt-induced oxidative stress when they were applied exogenously (**Table 6**). However, these are mostly dose dependent. A number of studies have been reported the positive effects of AsA in mitigating salt stress in wheat. Athar et al. [20] studied the effect of AsA on wheat plants subjected to salt stress. Salt stress (150 mM NaCl) caused reduction in growth and pho-

**Plant nutrients Protective effects References**

• Increased root length and biomass • Increased plant height and biomass

• Increased plant height and biomass • Increased chl *a*, chl *b* and carotenoid

• Increased SOD and POD activity

• Increased plant height, root length, root and shoot biomass

• Increased leaf number, leaf area and

• Decreased Na uptake and increase

• Increased root and shoot biomass • Increased root and leaf RWC • Increase K and Ca uptake

> •– and H2 O2 contents

• Decreased lipid peroxidation, electro-

• Increased SOD, POD, and CAT

uptake and decreased

Kausar and Gull

El-Lethy et al. [109]

Khan et al. [111]

Zaman et al. [112]

Tian et al. [113]

[110]

• Increased K+

content

chl content

K uptake

• Decreased O2

lyte leakage

activities

Na+ uptake

and moderately tolerant varieties. However, root applied AsA (100 mg L−1) counteracted the adverse effects of salt stress on the growth of tolerant variety which was due to the

/Na+

ratio in both sensitive

tosynthesis which were associated with decrease in tissue K+



**Table 6.** Protective effects of various exogenously applied antioxidants under salt stress in *T. aestivum*.

#### **4.5. Signaling molecules**

**Cultivars Dose and duration of stress**

168 Wheat Improvement, Management and Utilization

Sids 1 and Giza

168

S-24 and MH-97 150 mM NaCl, 58 d 50, or 150 mg L−1

3000 and 6000 mg L−1 NaCl,

throughout the growth duration

75 d

S-24 and MH-97 120 mM NaCl,

S-24 and MH-97 150 mM NaCl,

Waha 150 mM NaCl,

Giza 168 0.23, 3.85, and 7.69

Giza 168 0.8, 7.5, and 11.5 dS

75 d

4 weeks

2 weeks

dS m−1 salinity,

m−1 salinity, 65 d

Huaimai 17 300 mM NaCl, 7 d 100 μM SNP

AsA

200-600 mg L−1 ascorbin (ascorbic acid and citric acid 2:1)

50 and 100 mg L−1

100 mg L−1 AsA or α-tocopherol

(sodium nitroprusside, a nitric oxide/NO donor)

AsA

**Antioxidants Major effects References**

• Improved photosynthesis • Increased AsA content and CAT activities • Improved growth

• Decreased MDA content • Decreased activities of antioxi-

dant enzymes

POD, and SOD

• Decreased Na+

machineries

contents

• Decreased H2

500 μM melatonin • Improved shoot height, number

of shoot

pigments

dant activity

activities • Reduced H2

• Decreased Na+

activities

• Improved germination • Deceased Na content and increased K content • Enhanced CAT and SOD

• Lower Na+

0.7 mM AsA • Increased leaf area

• Improved photosynthesis

• Protection of photosynthesis

• Improved chl and carotenoid

• Enhanced Pro accumulation

• Increased photosynthetic

• Increased carbohydrate, protein, N, P, K, flavonoids, phenolic contents, and antioxi-

• Enhanced antioxidant enzymes

O2

lipid peroxidation, and membrane permeability

accumulation,

contents

and Cl<sup>−</sup>

O2 content

of leaves per plant, FW and DW

content

accumulation

100 mg L−1 AsA • Increased activities of CAT,

content, and

Athar et al. [20]

Elhamid et al. [114]

Athar et al. [115]

Khan et al. [116]

Azzedine et al. [21]

Sadak et al. [117]

Farouk [118]

Zheng et al. [119]

and Ca2+ content

• Decreased Na+

increased K+

Although there are specific signaling roles of phytohormones and antioxidants present in plants, which have been discussed in previous sections, this part will discuss the role of exogenously applied signaling molecules. Among the signaling molecules, nitric oxide (NO) has been widely studied in recent decades, due to its diverse role in tolerance to several abiotic stresses including salinity. Nitric oxide exerts its signaling role through various pathways and through interaction with other molecules (**Figure 3**) [26]. In the last decade, exogenous application of NO through different donors was found to enhance crop growth and productivity under stressful conditions [26]. Zheng et al. [119] observed great improvement in seed germination of wheat under high salinity (300 mM NaCl). Wheat seeds soaked in SNP solution provided better germination under salinity which was associated with decreased Na<sup>+</sup> concentration and increased K+ concentration in the seeds. Exogenous SNP also helped in increasing starch and amylase content in seeds which increased the weights of coleoptile and radical. Moreover, exogenous NO enhanced the activities of SOD and CAT which decreased the oxidative damages evident with lower level of lipid peroxidation, O2 •−, and H2 O2 [119]. Kausar and Shahbaz [120] found the positive effect of foliar applied NO in mitigating salt stress in wheat. Wheat seedlings grown under 100 mM NaCl exhibited reduced growth and photosynthetic rate. However, NO spray ameliorated the effect by enhancing FW of plants, leaf area, stomatal conductance, and internal CO2 concentration. However, NO could not take part role in enhancing PS II activity [120]. In our laboratory, we examined the effect of exogenous NO in conferring salt stress tolerance in wheat [24]. Wheat plant exposed to any level of salt (150 and 300 mM NaCl) caused significant increase in oxidative stress (as indicated by MDA and H2 O2 content). Salt stress-induced oxidative stress was due to the disruption of antioxidant defense. However, the seedlings which were pretreated with NO donor (1 mM SNP) showed enhanced tolerance which was due to increased nonenzymatic antioxidants (AsA and GSH pool) and the activities of monodehydroascorbate reductase (MDHAR), DHAR, GR, glutathione *S*-transferase (GST), GPX, glyoxalase (Gly) I, and Gly II. Therefore, we concluded that both antioxidant defense and glyoxalase systems worked together in enhancing salt stress tolerance as induced by NO [24]. As shown in **Figure 3** Arg is one of the precursors of NO production. Few studies have indicated the role of exogenous Arg in salt stress tolerance in wheat. Qados et al. [121] observed that Arg could alleviate the salt-induced adverse effects in wheat. When wheat plants were exposed to different levels of salinity (2000–8000 ppm NaCl), plant mass, relative water content, yield components (spike length, spike weight, and spikelets per spike), grain yield, straw yield, biological yield, and harvest index decreased in dose dependent manners. Salt stress also deteriorated the chemical constituents of the grains. However, when the grains were presoaked with Arg, they provided better growth, yield components, yield as well as the quality aspects (nutrient content) at harvest [121]. Polyamines are often considered as signaling molecules which interact with NO and also exert direct beneficial effects [124–126]. Saeidnejad et al. [122] found the positive effect of spermine (Spm) in mitigating salt stress (100 and 200 mM NaCl) effect in wheat. In general, although seed priming with Spm showed a slight effect on germination process on both susceptible and tolerant cultivars, Spm application was an effective approach in salinity tolerance induction of wheat cultivars mostly through the activation of enzymatic antioxidants and increasing osmolytes production [122]. H2 O2 , which was previously thought to be a toxic substance and a major ROS recently been considered as signaling molecules. The double role of H2 O2 is now an interesting topic of research of many plant scientists. However, as exogenous application, most of the experiments were conducted using H2 O2 as priming agents or pretreatments rather than using as cotreatment. Signaling cross talk of H2 O2 with NO is also well established since last two decades [127]. Exogenous H2 O2 protected wheat plants from salt-induced damages by enhancing antioxidant defense as reported by Li et al. [123]. The seedlings supplemented with H2 O2 (0.05 μM) decreased the levels of MDA and O2 •−, which was associated with the increased activities of SOD, POD, CAT and APX and the concentration of GSH and carotenoid under salt stress (150 mM NaCl). Exogenous H2 O2 also increased plant height, shoot length, root length, and biomass under saline condition. The results were reversed when H2 O2 scavenger was used that indicated a clear role of H2 O2 in initiating its signaling role when applied at lower concentration [123].

 **Figure 3.** Interaction with PA, H2 O2 , and Arg during NO biosynthesis.

#### **4.6. Seed priming**

tration and increased K+

170 Wheat Improvement, Management and Utilization

H2 O2

using H2

H2 O2 O2

the levels of MDA and O2

O2

O2

cross talk of H2

Exogenous H2

clear role of H2

stomatal conductance, and internal CO2

signaling molecules. The double role of H2

O2

concentration in the seeds. Exogenous SNP also helped in increasing

•−, and H2

concentration. However, NO could not take part role

O2

[119]. Kausar

O2

(0.05 μM) decreased

is now an interesting topic of research of many

O2

scavenger was used that indicated a

, which

starch and amylase content in seeds which increased the weights of coleoptile and radical. Moreover, exogenous NO enhanced the activities of SOD and CAT which decreased the oxi-

and Shahbaz [120] found the positive effect of foliar applied NO in mitigating salt stress in wheat. Wheat seedlings grown under 100 mM NaCl exhibited reduced growth and photosynthetic rate. However, NO spray ameliorated the effect by enhancing FW of plants, leaf area,

in enhancing PS II activity [120]. In our laboratory, we examined the effect of exogenous NO in conferring salt stress tolerance in wheat [24]. Wheat plant exposed to any level of salt (150 and 300 mM NaCl) caused significant increase in oxidative stress (as indicated by MDA and

activation of enzymatic antioxidants and increasing osmolytes production [122]. H2

as reported by Li et al. [123]. The seedlings supplemented with H2

saline condition. The results were reversed when H2

was previously thought to be a toxic substance and a major ROS recently been considered as

plant scientists. However, as exogenous application, most of the experiments were conducted

CAT and APX and the concentration of GSH and carotenoid under salt stress (150 mM NaCl).

protected wheat plants from salt-induced damages by enhancing antioxidant defense

as priming agents or pretreatments rather than using as cotreatment. Signaling

with NO is also well established since last two decades [127]. Exogenous

also increased plant height, shoot length, root length, and biomass under

O2

in initiating its signaling role when applied at lower concentration [123].

•−, which was associated with the increased activities of SOD, POD,

O2

 content). Salt stress-induced oxidative stress was due to the disruption of antioxidant defense. However, the seedlings which were pretreated with NO donor (1 mM SNP) showed enhanced tolerance which was due to increased nonenzymatic antioxidants (AsA and GSH pool) and the activities of monodehydroascorbate reductase (MDHAR), DHAR, GR, glutathione *S*-transferase (GST), GPX, glyoxalase (Gly) I, and Gly II. Therefore, we concluded that both antioxidant defense and glyoxalase systems worked together in enhancing salt stress tolerance as induced by NO [24]. As shown in **Figure 3** Arg is one of the precursors of NO production. Few studies have indicated the role of exogenous Arg in salt stress tolerance in wheat. Qados et al. [121] observed that Arg could alleviate the salt-induced adverse effects in wheat. When wheat plants were exposed to different levels of salinity (2000–8000 ppm NaCl), plant mass, relative water content, yield components (spike length, spike weight, and spikelets per spike), grain yield, straw yield, biological yield, and harvest index decreased in dose dependent manners. Salt stress also deteriorated the chemical constituents of the grains. However, when the grains were presoaked with Arg, they provided better growth, yield components, yield as well as the quality aspects (nutrient content) at harvest [121]. Polyamines are often considered as signaling molecules which interact with NO and also exert direct beneficial effects [124–126]. Saeidnejad et al. [122] found the positive effect of spermine (Spm) in mitigating salt stress (100 and 200 mM NaCl) effect in wheat. In general, although seed priming with Spm showed a slight effect on germination process on both susceptible and tolerant cultivars, Spm application was an effective approach in salinity tolerance induction of wheat cultivars mostly through the

dative damages evident with lower level of lipid peroxidation, O2

Seed priming is one of the easiest and cheapest techniques for successful crop production under various abiotic stress conditions including salinity [128, 129]. Seed priming is a presowing, controlled hydration technique that regulates and increases pregermination metabolic activity during early germination stage, but before radical projection [130, 131]. Seed priming has been effectively affirmed to improve germination percentage and seedling establishment in many crops such as wheat, rice, maize, soybean, canola, sunflower, sugarbeet, etc. [29, 132, 133]. Positive effects of seed priming might originate from de novo synthesis of certain germination-promoting substances, enhancing pregermination metabolites [131], early DNA replication, greater ATP availability, enzyme activation, osmotic adjustments [134], and membrane reorganization through restoring their original structures and reducing leakage of metabolites. Along with synchronous and fast emergence, primed seeds show reduced photo and thermodormancy, a wider range of germination temperatures and better capacity to compete with weeds and pathogens [135, 136]. Seed priming can be an easy solution for crops to overcome adverse environmental situations; it is reliable, simple, low cost, and also low risk technique [128, 137]. Various priming techniques such as hydropriming (soaking seed in water), osmopriming (soaking seed in nutrient, hormone, or chemicals), and halopriming (soaking seed in salt solution) have been developed to increase speed of germination, uniform seedling establishment, and crop production [138].

Seed priming has been effectively shown to increase germination and emergence of seeds of many crops in the tropical and subtropical areas, especially under salt stress conditions [139]. Increased germination rates and better seedling establishment resulted in higher levels of salt stress tolerance and crop yields when seeds were primed. Seed priming has recently been applied to overcome the salt stress problem on agricultural land [137]. Several research findings evidenced the role of seed priming to improve salt stress tolerance in wheat (**Table 7**). Hydropriming for 12 h on six Indian wheat cultivars showed 50% reduction of mean germination time under saline condition [140]. Effect of hydropriming was studied in salt-sensitive (MH-97) and salt-tolerant (AUQAB-2000) cultivars of wheat under salt stress (15 dS m−1) condition [16]. It is well documented that seed osmopriming helps to improve salt stress tolerance in wheat seedlings. Seed osmopriming with PEG-8000 solution showed increased germination percentage, germination index, root and shoot length, and seedling FW and DW than salt-affected wheat seedlings at different salinity levels (4, 8, 12, and 16 dS m−1). It has been reported that seed osmopriming with AsA helped to increase the endogenous AsA content and CAT activity which increased the salt stress tolerances in wheat [141]. Increased germination percentage, early seedling establishment, accumulation of ABA and Pro, and plant growth were featured due to seed osmopriming with 0.05 mM SA in wheat under salt stress condition [142]. Seed halopriming improves plant salt-tolerance by maintaining ion homeostasis mechanism. Salt stress increases the accumulation of Na+ concentrations in the roots and shoots of wheat plants and decreases the uptake of beneficiary nutrients. However, seed halopriming helps to maintain the ion homeostasis by decreasing Na+ concentration and increasing K+ , Ca2+ concentration, and K+ /Na+ ratio in roots and shoots. Increasing K+ and Ca2+ absorption, K+ /Na+ ratio due to seed halopriming under salt stress was connected with vigorous seedling growth and crop production, increased photosynthetic activity, and reduced electrolyte leakage. Seed halopriming with CaCl2 helps in the maintenance of ionic balance by reducing the Na+ and increasing the K+ absorption consequently improves salt stress tolerances [143]. Salt stress also induced oxidative damage by producing ROS. Seed halopriming detoxifies the ROS by increasing the activity of enzymatic antioxidant such as SOD and CAT [43]. Iqbal and Ashraf [100] demonstrated that halopriming with 100 mM KCl, NaCl, and CaCl2 reduced the salt stress affect on growth and grain production of two wheat cultivars. Priming with phytohormone increased germination with better seedling establishment and tolerance to various stresses including salinity. Seed priming of wheat with IAA increased germination percentage by improving amylase activity [144] and mitigated the growth inhibitory effect of salinity [16]. Seed priming of three wheat cultivars with auxin (0, 1, and 2 mg L−1) increased germination percentage, root and shoot length, seedling FW and DW, and yield under salt stress condition [18]. Priming with SA (100 mg L−1) solution for 24 h enhanced growth, photosynthetic pigments such as chl *a*, chl *b* and also increased total soluble and reducing sugar for maintaining osmotic adjustment during salt stress [145]. Iqbal and Ashraf [101] reported that seed priming with GA (150 mg L−1) played a potential role in alleviating salt stress damages by reducing Na+ and Cl<sup>−</sup> concentrations, Na+ / K+ ratio, and increasing K+ and Ca2+ contents. Moreover, seed priming with GA increased germination percentage, seedling growth and yield contributing components under salt stress condition.


Seed priming has been effectively shown to increase germination and emergence of seeds of many crops in the tropical and subtropical areas, especially under salt stress conditions [139]. Increased germination rates and better seedling establishment resulted in higher levels of salt stress tolerance and crop yields when seeds were primed. Seed priming has recently been applied to overcome the salt stress problem on agricultural land [137]. Several research findings evidenced the role of seed priming to improve salt stress tolerance in wheat (**Table 7**). Hydropriming for 12 h on six Indian wheat cultivars showed 50% reduction of mean germination time under saline condition [140]. Effect of hydropriming was studied in salt-sensitive (MH-97) and salt-tolerant (AUQAB-2000) cultivars of wheat under salt stress (15 dS m−1) condition [16]. It is well documented that seed osmopriming helps to improve salt stress tolerance in wheat seedlings. Seed osmopriming with PEG-8000 solution showed increased germination percentage, germination index, root and shoot length, and seedling FW and DW than salt-affected wheat seedlings at different salinity levels (4, 8, 12, and 16 dS m−1). It has been reported that seed osmopriming with AsA helped to increase the endogenous AsA content and CAT activity which increased the salt stress tolerances in wheat [141]. Increased germination percentage, early seedling establishment, accumulation of ABA and Pro, and plant growth were featured due to seed osmopriming with 0.05 mM SA in wheat under salt stress condition [142]. Seed halopriming improves plant salt-tolerance by maintaining ion homeostasis mechanism. Salt stress increases the

concentrations in the roots and shoots of wheat plants and decreases

helps in the maintenance of ionic balance by reducing the Na+

absorption consequently improves salt stress tolerances [143]. Salt stress

and Ca2+ absorption, K+

and Cl<sup>−</sup>

and Ca2+ contents. Moreover, seed priming with GA increased

, Ca2+ concentration, and

concentrations, Na+

ratio due to

and

reduced

/

/Na+

the uptake of beneficiary nutrients. However, seed halopriming helps to maintain the ion

seed halopriming under salt stress was connected with vigorous seedling growth and crop production, increased photosynthetic activity, and reduced electrolyte leakage. Seed

also induced oxidative damage by producing ROS. Seed halopriming detoxifies the ROS by increasing the activity of enzymatic antioxidant such as SOD and CAT [43]. Iqbal and

the salt stress affect on growth and grain production of two wheat cultivars. Priming with phytohormone increased germination with better seedling establishment and tolerance to various stresses including salinity. Seed priming of wheat with IAA increased germination percentage by improving amylase activity [144] and mitigated the growth inhibitory effect of salinity [16]. Seed priming of three wheat cultivars with auxin (0, 1, and 2 mg L−1) increased germination percentage, root and shoot length, seedling FW and DW, and yield under salt stress condition [18]. Priming with SA (100 mg L−1) solution for 24 h enhanced growth, photosynthetic pigments such as chl *a*, chl *b* and also increased total soluble and reducing sugar for maintaining osmotic adjustment during salt stress [145]. Iqbal and Ashraf [101] reported that seed priming with GA (150 mg L−1) played a poten-

germination percentage, seedling growth and yield contributing components under salt

Ashraf [100] demonstrated that halopriming with 100 mM KCl, NaCl, and CaCl2

concentration and increasing K+

accumulation of Na+

halopriming with CaCl2

ratio, and increasing K+

stress condition.

increasing the K+

K+ /Na+

K+

homeostasis by decreasing Na+

172 Wheat Improvement, Management and Utilization

ratio in roots and shoots. Increasing K+

tial role in alleviating salt stress damages by reducing Na+

• Increased SOD, CAT, APX activities



**Table 7.** Beneficial effects of seed priming in improving salt stress tolerance in *T. aestivum*.
