**3. Mechanisms of salt tolerance in oilseed** *Brassica*

To overcome salinity-induced osmotic and ionic stress, plants evolve avoidance or tolerance mechanisms in order to protect the cellular components from sustaining the growth and development. Ion homeostasis, vacuolar compartmentalization, accumulation of secondary metabolites, hormonal regulation, osmolytes production, and by activating defensive responses, plants endure the salinity-induced damages and enhance the stress tolerance.

### **3.1 Screening of salt-tolerant traits**

Screening of salt-tolerant cultivars of *Brassica* sp. is one of the most effective approaches to minimize the loss of yield; therefore, it has been attracted by many researchers and plant breeders. While working with 25 Indian *B. juncea* genotypes, Sharma et al. [99] reported that the lowest reduction of germination and speed of germination was found in the RB-10 and PR-2004-2 genotypes. Besides, the highest salt tolerance index for root growth was observed in six genotypes of mustard, such as RB-10, SKM-450, RK-05-02, JGM-03-02.RL-2047, and NRCD-509, and salt tolerance index for the dry matter was also highest in the RB-10 and PR-2004-2 genotypes. Finally, based on the results of germination, growth, and salt tolerance index, Sharma et al. [99] proposed that among the 25 genotypes, highly tolerant genotypes were RB-10 and PR-2004-2, whereas GN-48, JKMS-2, SKM −450, and CS-610-5-25P were categorized as tolerant and NDR-05-01, PBR 300, RK-05-01, NPJ-93, PDR-1188, and RGN-145 were moderately tolerant to salinity. Similarly, Yousuf et al. [39] experimented with 25 genotypes of *B. juncea* and reported among the 25 genotypes highest lipid peroxidation and lowest soluble protein content, antioxidant activities, and biomass accumulation were found in the Pusa Agrani genotype in a dose-dependent manner of salinity. Whereas, in CS-54 genotype salinity least affected the biomass accumulation, antioxidant activities together with minimal oxidative damages suggesting that CS-54 was more tolerant and the Pusa Agrani was sensitive genotype [39]. While working on the 21 genotypes of *B. juncea*, Prasad et al. [100] found the highest GP and vigor index in CS2009–347, followed by CS-52 genotype, and identified CS2009–347 and CS-52 as the most tolerant genotypes, whereas CS2009–256 and CS2009–145 genotypes were the susceptible genotypes under salinity. Previously, based on the P*n*, g*s*, T*r*, water use efficiency, C*i* and other physiological characters of 10 genotypes of *B. juncea*, Chapka Rohini was found to be the most susceptible to salinity, while Varuna was the most resistant genotype [101]. Moreover, Hossain et al. [13] studied the performance of four genotypes of *B. campestris* under salinity and found lower MDA, higher Pro, and antioxidants' activities in the salt tolerant genotypes (BJ-1603, BARI Sarisha-11 and BARI Sarisha-16) in comparison with the salt-sensitive genotype (BARI Sarisha-14).

#### **3.2 Osmoregulation**

To negate the cellular dehydration, plants must retort the osmotic balance, so it activates its osmoregulation mechanism to enhance salt tolerance [102]. In order to accomplish this, plants synthesize different compatible solutes or osmoprotectants such as Pro, glycine betaine (GB), sugars, trehalose, polyamines, organic acids, and amino acids to maintain the osmotic balance under salt stress. Besides osmotic balance, osmolytes are also engaged in ROS-scavenging, protect photosynthetic apparatus, maintain membrane integrity and protein stabilization [103]. In salttolerant cultivars of mustard, higher accumulation Pro was observed compared with the salt-sensitive cultivar [13]. Similarly, Ghassemi-Golezani et al. [104] found that accumulation of Pro and soluble sugars increased in a dose-dependent manner. So, based on the available literature, it can be stated that the accumulation of osmolytes helps to maintain osmotic adjustment and also induce tolerance in the salt-stressed *Brassica* plants (**Table 6**).

### **3.3 Hormonal regulation**

Hormones actively take part in the mediation and modulation of plant's responses to varying environmental conditions. The regulations of plant hormones are prominent in salt-stressed condition, and it can induce plant-adaptive mechanisms to cope with the stressed condition [106; **Table 7**]. Abscisic acid is a well-known stressresponsive plant hormone that helps in the mitigation of salt stress through increasing concentration within the plant to control stomatal closure and ultimately initiates


#### **Table 6.**

*Accumulation of osmolytes of oilseed Brassica sp. under salt stress.*

defense mechanisms. Upon exogenous application, ABA enhanced salt tolerance through attenuating the ionic and oxidative stress caused by salinity by lowering the accumulation of Na<sup>+</sup> and Cl− , and reducing the overproduction of H2O2 and TBRAS contents in *B. juncea* [74]. Additionally, the antioxidant activities of *B. juncea* were recorded to be increased even under salt stress due to ABA application as a consequence of increased activities of APX, GR, and SOD. Similarly, auxin, particularly indole acetic acid (IAA), plays an important role in the regulation of salt stress in *Brassica*. Being growth-generating hormone, auxin has the capacity to stimulate the growth attributes of plants, and this phenomenon also took place under salt stress in *Brassica* crops. Besides improving growth and photosynthetic characteristics including the recovery of stomatal aperture, the link between auxin and ROS resulted in well adaptation of *B. juncea* against salt stress [107] through uplifting enzymatic (CAT, POD, and SOD) and non-enzymatic (Pro) antioxidant activities.

Exogenous application of jasmonic acid (JA) increased ROS scavenging CAT, SOD, and POD activities with reduced TBARS content and thus, indicated amplification of salt tolerance of *B. napus* [110]. As a signaling molecule, ethylene (ETH) can modulate plant stress tolerance to some extent. Rasheed et al. [114] experimented that ethephon (an ETH-releasing compound) applied *B. juncea* plant resulted in better photosynthetic activities with improved stomatal behavior under salt-induced condition. With an increased APX, GR, and GSH activities and decreased H2O2 content within the plant, ethephon further strengthens the tolerance mechanism of *B. juncea* under the stressed condition imposed by salt. A similar role of ethephon was observed by Fatma et al. [45], whereas the elevated activity of AsA-GSH cycle to reduce the toxicity of H2O2 content in guard cells together with restricted ABA to initiate stomatal closure proved the salt tolerance mechanism of ethephon in *B. juncea.* Salicylic acid



**Table 7.**

*Hormonal regulation in salt stress tolerance of oilseed Brassica sp.*

(SA) is widely used in enhancement of crop stress tolerance and effective against salt stress too. Besides improving the physiological attributes of *B. carinata*, such as g*s*, P*n*, T*r*, and water use efficiency, SA can modulate TBARS and H2O2 contents to maintain the membrane stability under salt exposure [41]. Moreover, SA-treated *Brassica* plant can withstand salt-induced conditions due to the antioxidative mechanism of this hormone consisting of increased enzymatic (SOD, CAT, and POD) activities and ascorbate-glutathione pathway and that can ultimately maintain cell redox potential and ameliorate oxidative stress damage conferring salt tolerance ability of SA [41].

Brassinosteroids (BRs) can activate the stress-regulated genes and so take part in the stress amelioration of crops. In *B. juncea*, BRs in the form of 24-Epibrassinolide (24-EBL) showed better performance in bringing down the concentration of Na<sup>+</sup> , compliment with more K<sup>+</sup> and therefore, give rise to an increased plant height with higher fresh and dry biomass and improved Chl contents against salt stress [111]. The abatement of salt toxicity was further proved in that experiment by reduced endogenous ABA accumulation, EL, and lipid peroxidation together with uplifted GK and PROX activities that increase the Pro biosynthesis to combat the stressful condition in *B. juncea*. Retarded growth and quality of *B. nigra* were attenuated upon 24-EBL application with better antioxidant activities as 24-EBL activated antioxidant enzymes (SOD, POD), controlled MDA content, and encouraged the generation of secondary metabolites (phenolic and flavonoid contents) as well as anthocyanin content [104]. Moreover, the resistance potentiality of *B. juncea* after applying BRs has been reported as a consequence of adjusted ROS contents, uplifted antioxidant enzyme activities, and increased transcript gene (*BjAOX1a*) that cause elevation of a cyanide-resistant respiratory activity, to enhance the tolerance mechanism of *Brassica* in salt-induced condition [112]. Recovery of salt-induced stress by melatonin (MEL) has been proved in many kinds of research and so in oilseed *Brassica*. Like

other beneficial hormones, MEL is effective in amelioration of salt stress in *B. napus* growth, and in addition, MEL encourages the gene expression that linked with campesterol, JA, and GA hormones synthesis and properly regulates these hormones thus, ensured the salt tolerance mechanism of MEL [115]. Apart from this, root growth, which is the prime challenge under salt stress, was recorded to be uplifted after MEL treatment, resulted in increased root length, thickness, viability, and lateral root formation in *B. napus* due to the ability of MEL to impair the oxidative stress and maintain ion homeostasis [116].

### **3.4 Antioxidant defense**

To protect the cellular organelles from ROS-induced damages, plants are furnished with defensive mechanisms containing non-enzymatic and enzymatic antioxidants. In plants, non-enzymatic antioxidants such as AsA, GSH, flavonoids, and tocopherols, and enzymatic antioxidants such as SOD, APX, DHAR, MDHAR, GR, GST, glutathione peroxidase (GPX), and POD work in a coordinated manner in order to detoxify ROS [7]. In plant cells, SOD first activates, which converts O2 •− into H2O2, further transformation into less-reactive molecules take place in the presence of CAT, POD, GPX, or in the AsA-GSG cycle [117]. Under stressed conditions, the AsA-GSH cycle plays a crucial role in neutralizing H2O2 where AsA and GSH are accompanied by APX, DHAR, MDHAR, and GR in a cyclic manner [118]. Besides this, CAT, GST, GPX, polyphenols, and thioredoxins are also engaged in scavenging electrophilic substances, xenobiotics, and herbicides, and finally help in vacuolar transportation [119]. Plants are naturally equipped with the defensive mechanism to survive the stressed period by augmenting their activities. A number of papers have been published on the activities of antioxidant enzymes of *Brassica* sp. in salt-stressed conditions (**Table 8**). Upon exposure to salt stress (50 and 100 mM NaCl) to *B. juncea* cv. RGN-48, the activities of CAT, SOD, and POD are enhanced compared with the unstressed plants [15]. While working with four genotypes of *B. napus* (viz., BJ-1603, BARI Sarisha-11, BARI Sarisha-14, BARI Sarisha-16), Hossain et al. [13] found that activities of SOD, CAT, POD, and GPX were unchanged, whereas MDHAR and DHAR activities were decreased in the salt-sensitive cultivar (BARI Sarisha-14). On the contrary, antioxidant enzyme activities were increased in the salt-tolerant genotypes (BJ-1603, BARI Sarisha-11, BARI Sarisha-16) of mustard [13]. Another study from Husen et al. [41] found elevated activities of SOD, CAT, and POD in both cultivars (Adet and Merawi) of *B. carinata* upon exposure to salt stress, but in cv. Adet, the CAT and POD activities were higher, while activity SOD was more in cv. Merawi.

## **3.5 Stress signaling**

A complex array of mechanisms between different intracellular components is involved in stress signaling comprising reception, transduction, and induction of stimuli (**Figure 2**). ROS was previously believed as toxic molecule, but nowadays, ROS plays the role of signaling cascades. ROS can activate mitogen-activated protein kinase (MAPKs) pathway, which regulates the ionic homeostasis and osmotic adjustments [118]. In a well-organized and sequential pathway, MAPK cascades activated where phosphorylation of MAPK kinase kinase (MAPKKK) took place and transformed into MAPK kinases (MAPKKs) and MAPKs. Thus, the MAPK cascades transfer the stimuli of any environmental stresses to the target proteins and finally enhance gene expression and stress adaptation [120]. Thus, to maintain the osmotic adjustment, MAPK receives and transduces specific signals for the activation of genes


#### **Table 8.**

*Hormonal regulation in salt stress tolerance of oilseed Brassica sp*.

to synthesize osmoprotectants, such as Pro, trehalose, and sugars, which are involved in ROS quenching, maintains membrane integrity, and stabilizes proteins by sustaining water transportation system [121].

Excessive Na<sup>+</sup> elevates intracellular Ca2+ in the cytosol and activates Ca2+ signaling cascades. Calcium-permeable channel OSCA1 found in the plasma membrane as a putative osmosensor under osmotic stress due to the loss of function mutant *osca1* thus enhanced Ca2+ signaling pathway. Besides this, antiporter KEA1/2 and KEA3 also

#### **Figure 2.**

*A schematic representation of mechanisms of stress signaling of plants under salinity. After sensing the stress stimuli, plants activate secondary messengers of mitogen activated protein kinase (MAPKs) pathway through perception and transduction, which regulates adaptive responses. Specific gene expression plays vital role in synthesizing osmolytes (proline, pro; trehalose; glucose), phytohormones (abscisic acid, ABA; salicylic acid, SA; gibberellins, GAs), regulates defensive responses of antioxidants (superoxide dismutase, SOD; ascorbate peroxidase, APX; monodehydroascorbate reductase, MDHAR; dehydroascorbate reductase, DHAR; glutathione reductase, GR), and transporters of salt overly sensitive (SOS) in inducing tolerance of plants against salinity.*

augmented the osmotic-stress-induced Ca2+ signaling cascades by exchanging K+ in the plastids [122, 123]. Furthermore, ionic-stress-induced Ca2+ signaling mediated Na<sup>+</sup> -occupied calcium-permeable channel where the mutant of monocation-induced Ca2+ increases 1, *moca1* hypersensitive to salinity and enhanced Ca2+ influx by controlling the Na+ transportation [124]. The plasma membrane receptor-like kinase FERONIA (FER), leucine-rich-repeat receptor kinase, and hydrogen-peroxideinduced Ca2+ increases 1 (HPCA1) also involved in the stabilization of plasma membrane by transmitting the stress stimuli, thus maintain cell wall integrity and stomatal closure under salinity [125, 126].

Along with the Ca2+ signaling cascades, the salt overly sensitive (SOS) pathway also plays a crucial role in alleviating the ionic toxicity through exporting Na+ from cytoplasm to apoplast. Under salt stress, Ca2+ sensors (SOS3/SCaBP8) received the signals and transferred them to the serine/threonine protein kinase (SOS2) where it is phosphorylated with the SOS1 and increased salt tolerance of plants through augmenting the Na+ /H+ exchange capacity [127, 128]. Besides, ROS signaling activates defensive responses, which enhances antioxidant activities and scavenges ROS, thus helps in protecting the intracellular molecules through maintaining redox homeostasis [129].

Under salinity, osmoregulation is properly maintained as a result of ROS-induced activation of MAPK, and the Ca2+ signaling regulates the closing and opening of stomata. Besides this, H2O2 also played a crucial role in signaling cascades. Being a part of oxidative metabolism, H2O2 interplayed in between other biomolecules, such as ABA, ETH, SA, and NO at nontoxic level [130]. Thus, it helps to regulate the stressed period and enhance the tolerance capacity of the plants. Likewise, hydrogen sulfide (H2S) is a small gaseous signaling molecule performed in traversing of the intra- and inter-cellular domains and regulates the redox homeostasis in plants under

### *Oilseed* Brassica *Responses and Tolerance to Salt Stress DOI: http://dx.doi.org/10.5772/intechopen.109149*

stress. Moreover, H2S inhibits the dynamic synchronization of antioxidant enzymes and NADPH oxidase activity and induces the tolerance against stress [131]. Another molecule is nitric oxide (NO), which has capacity to modulate reactive nitrogen species (RNS), alter protein activity, metal nitrosylation, GSH biosynthesis, formation of tyrosine nitration/peroxynitrite, *S*-glutathionylation and *S*-nitrosylation to augment the stress endurance of plants [132]. Biosynthesis of PAs is regulated with the enhanced activity of NO, where NO reduces the PA oxidase activity, thus inhibiting the breakdown of PA and helping to resist the salt stress [133]. Furthermore, CO also increased salt tolerance through the NO-mediated signaling pathway, where plasmamembrane-localized proton pump (H<sup>+</sup> -ATPase) and antioxidant activities enhanced the stress tolerance capacity of plants [134]. Phytohormones have an indispensable role in regulating and enhancing stress tolerance of plants. Under salt stress, ABA activates kinase cascade pathway and regulates gene expression, thus increases endogenous ABA levels, which leads to stomatal closure to maintain the water balance in plants and also enhance selective absorption of ions for transferring Na<sup>+</sup> from the cytoplasm to the vacuole [135]. Whereas, GAs works antagonistically with the ABA to regulate the germination of seeds by augmenting enzymes and H<sup>+</sup> -ATPase activity. Beside this, GAs also reduce g*s* and increase transpiration and water use efficiency of plants to sustain the salt stress [136]. Cytokinins (CKs) perform differential role in the plant growth and development includes cell division, chloroplast biogenesis, apical dominance, leaf senescence, vascular differentiation, nutrient mobilization, anthocyanin production, and also known to induce salt tolerance in plants [137]. Synthesis of BRs under salinity enhanced the stress tolerances by regulating ionic homeostasis and osmoregulation and also responsible for the translational change of the stressresponsive proteins through expressing the stress-responsive genes to regulate Na+ /H+ antiporters activity [138]. Further growth of plants is modulated by the enhanced activity of auxin, whereas ETH signaling, together with ROS, is liable for the AsA biosynthesis under salt stress [139]. There are many biomolecules involved in the stress signaling pathway to adapt to adversity. But the interaction of these biomolecules and cross talk among the components are complex and yet to be discovered to interpret the adaptation of plants under salinity.
