Salinity Tolerance in Legumes: Classical and Molecular Breeding Perspectives

Abstract

Legumes, essential for global nutrition, confront challenges like environmental stresses like extreme temperatures, drought, and salinity. Salinity, affecting 8.7% of the planet’s area, poses a severe threat to legume cultivation, impacting physiological functions, nutrient balance, and nitrogen fixation. This chapter comprehensively explores the complex responses of legumes to salt stress, highlighting adaptive mechanisms such as osmotic stress tolerance, ion exclusion, antioxidant regulation, and hormone modulation. The breeding strategies, including molecular techniques like QTL mapping, association mapping, and transgenics, offer promising solutions to enhance salt tolerance in legumes. The knowledge regarding salt tolerance breeding is well-documented in cereals but not in legumes, emphasizing the identification of genomic regions associated with tolerance and the effective utilization of molecular tools. Wild relatives provide valuable tolerance genes, requiring detailed understanding of their roles at different developmental stages. The multi-environment screening and integration of diverse breeding approaches, including genomics, transcriptomics, metabolomics, transgenics, and CRISPR-Cas9, is essential for developing legumes capable of thriving in saline environments and exhibiting high-salt tolerance.

1. Introduction

Legumes though cultivated in substandard arable soils, serve as a vital source of nutrition for billions of people across the globe and are a good source of protein, dietary fiber, carbohydrates, vitamins, and minerals. Globally, in an annum, legumes are grown in approximately 5.8% of gross cultivable poor land area [1]. In India, pulses are cultivated on around 28.7 million ha, with a production and productivity of about 25.4 MT and 885 kg/ha, respectively (www.indiastat.com). Various legumes viz., pigeon pea (Cajanus cajanifolius), black gram (Vigna mungo), green gram (Vigna radiata), chickpea (Cicer arietinum), soybean (Glycine max), field pea (Pisum sativum), cowpea (Vigna unguiculata), horse gram (Macrotyloma uniflorum), moth bean (Vigna aconitifolia), lablab bean (Lablab purpureus), lentil (Lens culinaris), French bean (Phaseolus vulgaris), and khesari (Lathyrus sativus) are grown worldwide as either sole or intercrop. Legume cultivation will also improve the soil structure and infiltration rates, but its production has fluctuated over the past few decades depending on environmental conditions [1]. Any changes from the ideal environmental conditions, whether it is an abundance or shortage, is considered abiotic stress, leading to negative impacts on the growth and development ultimately affecting the productivity of plants [2]. Various environmental stresses like soil salinity, extreme temperatures, and excess and deficit water available situations, have influenced the production of legumes. Salinity is considered the most perilous stress. Legumes are highly sensitive to salinity stress compared to cereals. When the concentration of salts in soil is too high, it can have negative impacts on plant growth, yield, and the physical properties of the soil [3]. Cultivating salt-tolerant plants is crucial for utilizing salt-affected agricultural land sustainably, maximizing land use, conserving resources, and enhancing resilience to climate change [4]. More than 833 million hectares of area around the globe are salinity-affected (8.7% of the planet) and majorly found across arid or semiarid environments of Asia, Latin America, and Africa [5]. The accumulation of salts predominantly occurs through natural processes over a prolonged period. For example, during the process of weathering, rocks discharge various soluble salts at different degrees. The major discharges are Na (sodium), Ca (calcium), and MgCl₂ (magnesium chloride) while carbonates and sulfates are released at a lower intensity. NaCl is the prevalent type among them. Rainwater also contains 10 mg/kg of sodium chloride, thus for every 100 mm of annual precipitation, it would leave behind 10 kg/ha of salt deposition in the land. Saline water irrigation influences the salt contents in the water table which negatively affects the crop performance [6]. Saline soils are characterized by a higher electrical conductivity (EC) i.e. more than 4 dS/m, which is approximately equal to 40 mM NaCl. The dominant ions found in such soils are Cl⁻, SO₄⁻, and in a few cases, NO₃⁻. Salinized areas are expanding by 10% each year due to multiple factors like insufficient rainfall, increased surface evaporation, natural rock erosion, inadequate agricultural practices, and improper irrigation using saline water. If these grave concerns continue, it is projected that more acreage of cultivatable land (>50%) will become saline in 2050 [7]. In India, 2.956 Mha is saline affected and 3.771 Mha is sodic soil which equates to 2.1% of its geographical area [8]. Sodic soil, on the other hand, is characterized by its bicarbonate content, high pH, and ESP values greater than 15. The characteristics of saline and sodic soils are furnished for a better understanding in Table 1. The complexity and presence of low genetic variability for salinity restrict the use of classical breeding techniques for developing salt-tolerant lines in legumes. Furthermore, the lengthy breeding cycles in classical breeding and other environmental influences hinder the progress in developing salt-tolerant crop varieties. Understanding the molecular mechanisms behind salinity will pave the way to genetically control or modify the genes responsible for salt tolerance in legumes, thereby enabling the development of salt-tolerant varieties through advanced breeding methods or genetic engineering techniques [9]. Genetic engineering facilitates the introduction of salt-tolerance genes into plants, enabling them to cope with high salinity levels in the soil. These genes typically encode proteins or mechanisms that regulate ion uptake and transport, maintain osmotic balance, or detoxify ions within plant cells, ultimately enhancing the plant’s ability to thrive in saline environments [10]. The omic techniques like transcriptomics, proteomics, and metabolomics, are valuable for comprehensively understanding salinity tolerance responses in legumes. It allows the researchers to understand and analyze the gene expression patterns, protein profiles, and metabolic processes of legumes under salinity conditions [11]. Transcriptomic studies analyze the expression levels of genes in plants exposed to abiotic stress, revealing key genes and pathways involved in stress responses. By identifying these genes, researchers can target them for genetic modification to enhance the plant’s ability to tolerate salinity conditions [12]. Both proteomics and metabolomics are advanced techniques that enable the comprehensive study of proteins and metabolites in plants [13]. During the salinity stress, each genotype will show differences in protein abundance and metabolite composition. Comparing the proteomic and metabolomic profiles of different genotypes under salt stress can elucidate common pathways or mechanisms involved in salt tolerance, facilitating the development of more tolerant genotypes [14]. In light of the above, this review aims to consolidate the facts of salinity, its ill effects on legume performance, legumes’ ability to withstand salinity stress, and the employable crop improvement strategies to combat salinity-induced stress.

Soil type	Electrical conductivity (EC)	pH	Exchangeable sodium percentage (ESP)	Impact on plant growth
Saline soils	≥4 dS/m	<8.5	<15	The growth of roots and shoots is impeded by stress caused by both osmotic and ionic factors
Sodic soils	˂4 dS/m	>8.5	≥15	Poor soil structure suppresses root growth

Table 1.

The characteristics of saline and sodic soils.

2. Effects of saline stress on legumes

Salt stress elicits a various response in agricultural crops. High salinity levels (HSL) are detrimental to plants. HSL affects crucial physiological functions, nutrient balance, hormonal regulation, and biological nitrogen fixation in legumes [15]. These alterations induce flower abortion, reduce carbon fixation, produce a few flowers and pods, & ultimately hinder crop production (Figure 1) Generally, salinity stress occurs in two phases viz., osmotic phase and ionic phase. During the initial stage, the presence of salt outside the plant’s root area can influence its reaction to salinity by restricting water absorption through roots, leading to reduced shoot growth, and delaying new leaf emergence and new lateral buds. Subsequently, in the later stage, the accumulation of salt within the plant tissues can cause toxic effects. It is evident that salinity affects every phase of plant growth, encompassing germination, vegetative growth, and reproductive growth [7]. However, it has a greater impact on seed germination and seedling growth compared to later stages of plant development [16]. During the germination stage, the rate of germination decreases, which negatively affects development of roots and plants establishment. This occurs when there are high levels of ions that hinder water absorption. As a result, both ion toxicity and osmotic stress are induced. At later stages of plant development, an excessive buildup of ions in the shoots hampers the process of photosynthesis, leading to premature aging of leaves. The inhibition of plant growth is frequently linked to a decline in photosynthetic activity. During the reproductive stage, due to nutritional imbalance, flower abortion will be more, leading to a reduced number of flowers and ultimately reducing the yield. Salinity reduces the number of pods and seed weight in soybean [17]. Similarly, Ghassemi-Golezani et al. [18] reported a reduction in protein and oil content. Mshelmbula et al. [19] evaluated the effect of salinity on the germination, growth, and yield performance of cowpea using different concentrations of NaCl. The NaCl stress reduces growth parameters and root nodules, due to an increased influx of toxic ions, and efflux of cytosolic solutes in plant cells. Nodulation is the imperative process in legumes which helps in fixing nitrogen required for the entire crop cycle. However, the mechanism of nodulation and nitrogen accumulation is altered by the salt stress in soybean and alfalfa [20]. Bolanos et al. [21] studied that the nodule weight and nodule number have decreased in pea plants exposed to salinity and many nodules appeared pale in color when compared to control. Nitrogenase enzyme activity in nodules can be quantified readily with the acetylene reduction method. Acetylene reduction activity (ARA) was significantly reduced when the Phaseolus vulgaris genotype was treated with 50 mM NaCl [22]. Additionally, nitrogen fixation and other antioxidant enzyme activity in nodules were also impacted. In summary, salinity negatively affects legumes by disrupting seed germination, photosynthesis, nutrient absorption, nutrient balance, and ultimately reducing crop yield.

Figure 1.

The impacts of salinity on legumes (created with https://www.biorender.com).

3. Tolerance mechanisms

Plants employ various strategies to thrive in saline conditions and those strategies can vary depending on the plant species. They can be either adaptive or specific mechanisms. The genetic control mechanisms linked with salinity tolerance are intricate and typically involve multiple genes and functions.

3.1 Osmotic stress tolerance

Osmotic stress occurs when there is a difference in salt concentration between the plant’s cells and the surrounding soil or water. This difference creates an imbalance of water, leading to a condition where water moves out of the plant’s cells, causing dehydration and impaired cellular functions. The reduction in turgidity modifies hormone levels and gene activity by elevating the presence of abscisic acid (ABA), resulting in enhanced osmotic regulation to mitigate the impact of salinity, thereby preserving the rigidity of leaves. Osmotic stress adjustment occurs through the uptake of a few ions and the synthesis of osmolyte solutes like sugars, amides, polyols, amino acids, etc. A list of osmolytes and their crucial role in salinity tolerance is listed in Table 2. Osmolytes are polar and soluble organic compounds that do not influence the functionality of cellular metabolic pathways [23]. Proline and Glycine betaine serve as crucial osmolytes that participate in osmoregulation, counteracting the detrimental effects of osmotic stress specifically in many grain legumes [24]. For instance, cysteine treatment reduces the adverse effect of salinity by raising the proline content, catalase and superoxide dismutase activities, and photosynthetic pigments [25]. The efficiency of proline and glycine betaine in increasing the salt tolerance in chickpea were investigated by Dawood et al. [26]. The results showed proline treatments (5 mM) were more effective than glycine betaine in increasing the salinity tolerance of chickpea.

Osmoprotectants/compatible solutes	Role in salinity stress tolerance
Proline	It acts as a free radical scavenger, balances cell redox levels of the cell and acts as a signaling molecule to coordinate mitochondrial function
Glycine betaine (GB)	Aids in osmotic adjustment and protecting the thylakoid membrane, which helps to maintain efficient photosynthesis.
Mannitol	Scavenging the free oxygen radicals
GABA	Helps in carbon-nitrogen balance and ROS scavenging and improves abiotic stress tolerance
Trehalose	Stabilizing the proteins and membranes during stress conditions. It is also involved in regulating carbohydrate metabolism.
Fructan	Scavenging ROS and osmotic adjustment

Table 2.

A list of osmolytes and their role in salinity tolerance.

3.2 Ion (Na+) exclusion

Salinity tolerance is the function of ion exclusion (Na⁺ and Cl⁻) and vacuole compartmentalization. The sodium ions reaching the cytoplasm are subsequently diverted to the vacuole with the help of a Na⁺/H⁺ antiporter. The ion compartmentalization and exclusion strategy will help in protecting the reproductive organs [6]. The salt overly sensitive pathway (SOS) (Figure 2) is the immediate ion homeostasis mechanism activated in plants upon sensing abiotic stress [27]. Precise regulation of intracellular sodium and potassium ion flux is essential for the optimal activity of diverse cytosolic enzymes, cell volume regulation, and for maintaining the membrane potential. The survival of plants under salinity stress is greatly influenced by the homeostasis between Na⁺ and K⁺. The SOS signaling pathway comprises SOS1, SOS2, and SOS3 proteins (Table 3), which play integral roles in the pathway’s functioning.

Figure 2.

SOS pathway (Salt Overly Sensitive Pathway) (created with https://www.biorender.com).

Proteins	Functions
SOS1	Encodes Na^+/H^+ antiporter (plasma membrane bound) Regulates sodium ion efflux, pH homeostasis, membrane vesicle trafficking, and vacuole functions and Enables long-distance Na^+ ion transport
SOS2	Encrypts a serine/threonine kinase
SOS3	It has a myristylation site at N-terminus, this site regulates salt tolerance

Table 3.

The proteins engaged in SOS pathway and their roles.

The overall function of this signaling pathway involves a cascade of events. When the concentration of Na⁺ increases, it leads to a rapid rise in intracellular Ca²⁺ levels, enabling its interaction with the SOS3 protein. This interaction, facilitated by Ca²⁺, regulates Na⁺ levels along with SOS proteins. Subsequently, SOS2 gets activated by the SOS3 proteins through stimulation of the kinase activity. This SOS3/SOS2 complex phosphorylates SOS1 upon transportation to the plasma membrane. This phosphorylation of SOS1 leads to an enhanced efflux of Na⁺ by stimulating its antiporter activity which ultimately reduces the Na⁺ toxicity.

4. Antioxidant regulation of salinity tolerance

Due to salinity stress, there will be a rapid accumulation of reactive oxygen species (ROS) in the cytosol. The predominant ROS are hydrogen-peroxide (H₂O₂), hydroxyl radical (^•OH), superoxide-radical (O₂^•−), and singlet-oxygen (O₂). Their production is cytotoxic but they also serve as signaling molecules that stimulate signal transduction pathways in response to stress [28]. Excessive ROS generation during stress will lead to the degradation of lipids in the cell membrane, proteins, and nucleic acids. However, salt-tolerant legumes possess their own antioxidant defense system, which helps scavenge ROS and protect cells from oxidative damage. Various enzymatic and nonenzymatic antioxidants involved in a scavenging activity are carotenoids, tocopherols, flavonoids, flavones, ascorbic acid, glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), glutathione peroxidases (GPX), superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) [24]. Thus, the oxidative stress caused by the ROS is tackled by these antioxidant activities.

5. Hormone regulation and salinity tolerance

Abscisic acid (ABA) is noticed as an immediate response to saline and drought stresses which subsequently triggers numerous adaptive responses in plants [27]. In legumes, ABA plays a major role in multiple stress responses, including metabolic alterations, closure of stomata, and activation/regulation of several stress-inducible genes thereby the correlated protein syntheses are regulated. Such a regulation helps the plants to adapt to the salinity stress through enhanced osmotic adjustment.

6. Screening methods for identification of salinity-tolerant genotypes/donors

To develop plant varieties that are tolerant to salinity stress, it is important to establish reliable screening methods that can effectively assess their tolerance to high-salt levels. Duration and growth stage are important factors for evaluation and screening for salinity tolerance [29]. Generally, salt stress is assessed by comparing productivity with saline and non-saline conditions. In the case of HSL, the survival capacity is often used as a reliable measure. It is considered that seedling screening for salinity tolerance is noteworthy as seedling establishment under salinity is the basic requirement for a better crop establishment and yield. Various selection criteria including seedling survivability score, assessing proline content, Na⁺, and Cl⁻ accumulation, and production of H₂O₂ can be used to assess the salt tolerance at seedling level. Rapid screening methods like the salinity induction response (SIR) technique and hydroponics are the commonly used techniques to identify putative donors in a breeding program.

6.1 Salinity induction response (SIR) technique

This technique was developed and standardized by the University of Agricultural Sciences, Bengaluru for various cereals and legumes [30]. In the salt induction and recovery (SIR) technique, young seedlings are first exposed to a mild level of salt stress, which serves as an initial “induction stress.” After this exposure, the seedlings are then subjected to a high concentration of salt for a particular duration. Following this period, the seedlings are transferred back to a normal water environment to allow for recovery. The extent of recovery growth observed in the seedlings is then used as a measure of their salt tolerance. Manasa et al. [30] screened 40 mung bean lines at the seedling level using SIR technique and identified a few lines with better biomass and pod yield than control.

6.2 Screening using hydroponics technique

Screening plants for salinity tolerance in field conditions can be challenging due to soil variations and environmental factors. As an alternative, laboratory-based techniques like hydroponics offer a more controlled environment with uniform salinity levels in the growth medium. However, to accurately assess plant traits, it is crucial to identify a specific stage in a plant’s growth cycle that is most sensitive to salt stress. In this technique, salt is added to the nutrient hydroponic solution [31] in which the seedlings are grown and scoring is done based on the percentage of leaves wilted. The scoring for assessing the salt tolerance level in seedlings is given in Table 4.

Percentage of leaves wilted	Scoring
76–100%	1
51–75%	3
26–50%	5
1–25%	7
Normal healthy leaves	9

Table 4.

Evaluation of salt tolerance levels in seedlings using assessment scores.

6.3 Salt tolerance index (STI) and Salt injury index

Under the salinity condition, STI is calculated as the ratio of the total dry weight of salt-stressed plants to their respective controls [32]. Salt injury index is calculated using the formula, (1-STI).

6.4 Physiological parameters

Plants exhibit remarkable physiological changes, including changes in signaling pathways and production of antioxidant enzymes, in response to varying levels of salinity which enable them to thrive in different saline conditions [33]. Thus, parameters like chlorophyll content, proline content, total sugars, production of H₂O₂, other antioxidant activities, and stomatal conductance at different growth stages serve as reliable measurements for evaluating salt tolerance in legumes.

7. Breeding strategies for enhancing the salt tolerance

Various breeding strategies offer promising solutions to overcome productivity losses due to salinity. The following sections highlight different breeding strategies and omics approaches that are employed to enhance salt tolerance in plants. Conventional breeding methods rely on the careful selection and crossbreeding of plant varieties with desirable salt tolerance traits. Through successive generations, these breeding programs gradually amplify the presence of salt-tolerant genes within plant populations. Additionally, selection and screening techniques enable the identification of salt-tolerant donors or germplasm collections which can serve as valuable genetic resources for breeding programs.

7.1 Mutation breeding

Mutagenesis involves inducing genetic mutations in plant populations, leading to a wide range of genetic variations. Some of those mutations may lead to enhanced salt tolerance. Through careful screening in salt-stressed conditions and selection of mutated individuals, breeders can identify and incorporate beneficial mutations into breeding programs. Thus, it facilitates the development of salt-tolerant varieties [29].

7.2 Assessing the genetic potential of wild relatives for salt tolerance

The existence of ample genetic variability is a crucial element for the successful advancement of crop improvement initiatives. Crop wild relatives are the potential genetic resources for crop improvement [34]. Many reports specify that they harbor resistance genes for various traits like disease, salinity, drought, and heat tolerance. Through various breeding approaches like hybridization or molecular techniques, these desirable traits can be introduced into cultivated species.

This transfer of traits from wild relatives enables the transfer of valuable genetic characteristics, such as disease resistance, tolerance to abiotic stresses, improved nutritional content, and overall agronomic performance, into cultivated crops. Some wild species harboring the salt-tolerant genes in legumes are listed in Table 5.

S. no	Crop	Wild species	Reference
01.	Red gram	C. acutifolius; C. sericeus	[35]
		C. scarabaeoides	[36]
		C. albicans: C. cajanifolius	[37]
		C. platycarpus	[38]
02.	Chick pea	C. microphyllum	[39]
03.	Cowpea	V. marina; V. luteola, V. vexillata	[40]
		V. trilobata; Vigna vexillata	[41]
		V. unguiculata subsp. sesquipedalis	[42]
04.	Soybean	G. soja	[43]

Table 5.

Sources of salinity-tolerant traits identified in wild species.

Linkage drag is an unexpected character transmission that is witnessed in most of the crop improvement programs involving wild species. Such linkage drags either restrict the direct utility of segregants or warrant employing other breeding strategies to overcome the issue of unwanted trait transmission. Evolution of introgression lines (ILs) with the targeted traits with a better yielding potential will increase the chance of utilizing the wild species in commercial plant breeding programs. The progress in genetic inheritance, evaluation methods, software tools, molecular markers, germplasm modification, and mapping has significantly contributed to the enhancement of salt tolerance and other abiotic stress traits.

8. Molecular and omics tools for salinity breeding

8.1 Genetic mapping and identification of QTLs associated with salt tolerance traits

Earlier genetic study indicates that the inheritance pattern on salinity tolerance is governed by many genes and is a complex quantitative trait [9]. The genetic basis and mechanism of these quantitative traits can be improved by identifying the genomic regions associated with the trait. The use of various DNA markers like random amplification of polymorphic DNA (RAPD), simple sequence repeats (SSR), and restriction fragment length polymorphism (RLFP), etc., relevant to several abiotic stresses has been reported in various legumes.

In chickpea, a RIL population was developed to map the salinity tolerance QTLs by utilizing two extreme parents ICCV 10 and DCP 92-3. The former genotype is saline tolerant and the latter is susceptible and identified 28 key QTLs [44]. An F6:F7 population of 97 RILs, obtained from a cross between faba bean (Vicia faba L.) cultivars Icarus and Ascot, was utilized for QTL mapping and identified seven QTLs associated with salt tolerance [45]. Atieno et al. [46] developed an RIL population by hybridizing Genesis836 and Rupali chickpea varieties using DArT and SNP markers. The former genotype is saline tolerant and the latter is susceptible. A total of 21 QTLs controlling various yield-related traits under salinity were found on CaLG04. In soybean, on chromosome 3, a major QTL flanked by Barcsoyssr_03_1421 and GMABAB (SSR markers) conferring saline tolerance was reported by Shi et al. [47]. This marker trait-associated information can be utilized to enhance genetic gain in the salt-tolerance breeding programs in legumes.

8.2 Association mapping (AM)

AM is the high-resolution method for mapping QTL that relies on the principle of linkage disequilibrium (LD) [48]. AM utilizes GWAS with SNPs and candidate genes analysis (CGA) for mapping. GWAS utilizes the genetic variation of a population (involving many individuals) to assess the association between genotype and phenotype(s). While, in CGA, markers are selected either based on their genomic location or based on published QTL studies. Ravelombola et al. [49] evaluated salt tolerance in cowpea MAGIC population (involving 234 lines) using GWAS, and identified a large variation for salt tolerance conferring traits. Candidate genes related to Na⁺/Ca⁺, K⁺ independent exchanger, and salt tolerance gene Vigun01g093100.1, calcium-dependent protein kinases were identified. Nine candidate genes were identified in soybean during seed germination in response to salt stress. Of them, Glyma08g09730.1, Glyma08g12400.1, Glyma18g47140.1, Glyma09g00490.3, and Glyma09g00460.1 were verified with respect to salt stress [50]. In the mungbean germination stage, Breria et al. [51] used GWAS approach with 5288 SNP markers to understand the QTL(s) responsible for salt tolerance. Significant SNPs associated with salt tolerance were identified at chromosomes 7 and 9 containing genes Vradi07g01630 (ammonium transport protein), Vradi09g09600, and Vradi09g09510. A total of 283 soybean lines were used for the GWAS study using the SoySNP50K chip Zeng et al. [52] showed nine genomic regions corresponding to leaf chloride and chlorophyll concentrations.

8.3 Omics tools for salinity breeding

8.3.1 Transcriptomics

RNA-seq has been widely used for studying the differential expression of genes. It provides information related to allele-specific expression, isoforms, and other novel/key promoters which are not possible through transcriptomic studies [53]. During salinity stress, the control of gene expression is influenced by different pathways involving many transcription factors (TFs) like DREB, bHLH, AP2/ERF, bZIP, GATA, Homeo-box, HD-Zip, MADS-box, Trihelix, MYB, WOX, NAC, YABBY, WHIRLY, zinc finger, and WRKY [54]. These TFs play a significant role in ROS production, chlorophyll content, and lipid peroxidation. Thus, they enable the plants to withstand unfavorable conditions and regulate the developmental process in response to abiotic stress [12]. Zeng et al. [55] investigated the RNA-Seq data of the RA-452X Osage population of soybean and revealed numerous genes that exhibited differential expression in the salt-tolerant line. Through gene annotation, they identified several key potential genes (Glyma.04G180400, Glyma.02G228100, Glyma.03G226000, Glyma.03G031400, Glyma.03G031000, Glyma.04G180300, Glyma.05 g204600, Glyma.17G173200, Glyma.08G189600, and Glyma.13G042200) involved in tolerance mechanism. Similarly, Jia et al. [56] explained the salt tolerance mechanism of f-box genes in soybean by analyzing 12 salt-responding F-box genes. Kaashyap et al. [57] studied the effect of differentially expressed genes in chickpea indicating various salt stress response genes like thaumatin, enzyme inactive 2- (AOP2), jasmonic acid-amido synthetase (JAR1), aminocyclopropane-1-carboxylate oxidase, potassium transporter gene playing a crucial role in enhancing salt tolerance. Many genes are transcriptionally activated in response to salinity, resulting in the production of altered metabolic proteins under salt stress conditions. These proteins play a regulatory role in downstream gene expression and enhance stress tolerance mechanisms [13]. Under salinity stress in common beans, PvbHLH genes (63 Nos.) were expressed differentially. Among that, the participatory role of PvbHLH-54 and PvbHLH-148 under salt stress was evaluated using qRT-PCR [58]. Likewise, in the case of chickpea [59], the expression levels of WRKY-TF genes were found to be differentially regulated in response to salinity stress. In functional nodules of soybean under salt stress, Dong et al. [60] examined the dynamic regulation of miRNAs. They discovered eight potential miRNAs associated with the stress signaling pathway, ion and osmotic balance, and ABA signaling. Spatial transcriptomics and functional profiling help in identifying differentially expressed genes and pathways and uncover spatially regulated biological processes by integrating both mRNA expression data with corresponding histological information of the tissue [61]. This is an advanced technique that combines traditional RNA sequencing with spatial information to study gene expression within the context of tissue architecture. While conventional transcriptomics provides gene expression data from bulk samples, spatial transcriptomics allows researchers to analyze gene expression patterns within the spatial context of tissues or organs. It enables the generation of spatially resolved transcriptomic data, providing valuable information about the location of specific gene expression within a tissue or organ. Employing this technique will greatly advance our understanding of salinity tolerance in legumes.

8.3.2 Proteomics

Proteomics provides insights into the dynamic protein changes associated with fundamental biological pathways as well as the posttranslational modifications of proteins induced during stress. These modifications are critical for plants to effectively adapt and acclimatize to diverse abiotic stresses [62]. Proteomics helps to unravel the strategies employed by plants at the cellular and tissue level to thrive under salt stress. Also, it facilitates the identification of key genes and proteins associated with salt tolerance. Differentially expressed proteins studied in different legumes are listed in Table 6. These genes are cloned and introgressed/transferred to various salt-sensitive genotypes. Qiu et al. [68] identified GsMAPK4 protein kinases are the positive regulatory factors for salt tolerance in soybean using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). In the same way, ROS scavenging and photosynthetic capacity of GsCBRLK proteins in response to salt stress were studied using an iTRAQ-based proteomic approach in soybean by Ji et al. [14]. Proteins like rubisco activase, Ru5PK (ribulose-5-phosphate kinase), and OEE (oxygen-evolving enhancer) protein 2, are expressed in salt-tolerant cowpea genotypes indicating their integral roles in energy metabolism and photosynthesis [69].

Crop	Proteomic technique	No. of identified proteins	Major findings	Reference
Soybean	iTRAQ	278	Proteins related to stress signal transduction and membrane proteins were upregulated which leads to ROS scavenging and tolerance to other biotic as well as abiotic stresses including salinity tolerance.	[63]
Chickpea	LC-MS/MS	364	Salinity-tolerance-associated proteins like chlorophyll a-b binding protein, ATP synthase, LEA, ascorbate peroxidase, and ribonucleoproteins are the key regulators of salt-response, signaling, and energy metabolism.	[64]
Alfalfa	iTRAQ-LC-MS/MS	438	The salt tolerance mechanism depends on the proteins associated with antioxidants, detoxification enzymes, glutathione metabolism, and secondary metabolism.	[65]
Cowpea	MALDI-TOF/TOF mass spectrometry	5	Up-regulation of pentatricopeptide repeat protein, flavanone 3-hydroxylase ATP synthase, vacuolar ATPase, and outer-envelope pore protein enhance salt tolerance. These proteins are mainly related to energy metabolism, ion homeostasis, defense, and transport of ions.	[66]
Soybean	LC-MS/MS	972	Differentially expressed proteins in leaves and roots include proteins linked to cation and anion channels, calcium-sensing proteins, receptor kinases, abscisic acid receptors, and flavoprotein oxidoreductases confirming their involvement in salt stress tolerance.	[67]

Table 6.

Differentially expressed proteins studied in different legumes.

8.3.3 Metabolomics

Metabolomics refers to the analysis of metabolites for the evaluation of biological reactions triggered by abiotic stresses [62]. The metabolic responses are important indicators for evaluating salt stress tolerance in crop species. Salt stress triggers osmotic imbalances, leading to harmful changes at physiological and molecular levels in cellular components. To overcome the salt-induced osmatic imbalance and other associated ill effects, plants produce/modify a variety of metabolites. The chemical complexity and dynamic nature of metabolites limit the metabolomic platforms to profile all the metabolites at a given time [70]. Numerous studies utilizing metabolomic analyses have been carried out to examine the underlying mechanisms for salinity stress tolerance (Table 7). Metabolomic techniques like NMR spectroscopy and mass spectrometry (MS) have greatly aided in studying the metabolite profiles of plants and understanding their mechanisms of stress tolerance. The production of various metabolites including proline, lactose, ribose, lauric acid, palmitic acid, stearic acid, linolenic acid, mucic acid, glutaric acid, galactonic acid, and dehydroascorbic acid demonstrated the higher salinity tolerance in wild soybean [71].

Class of metabolites	Metabolite	Plant species	Function	Reference
Amino acids	Valine, tyrosine, glutamic acid, leucine, and isoleucine	Soybean	Stabilizing the intracellular pH and balancing osmotic pressure.	[71]
Polyamines (PA)	Putrescine, spermidine, and spermine	Common beans	Maintenance of ion levels, counteracting free radicals, membrane stabilization, and protection of cellular structures.	[72]
Sugars	D-trehalose	Lentil	Osmo protectant	[73]
Amino acids	GABA	Green gram	Improves growth, photosynthesis, enzymatic and nonenzymatic antioxidative defense mechanisms, and nitrogen metabolism during salinity stress.	[74]
Sugar alcohol	d-arabitol	Soybean	Osmo protectant	[75]
TCA cycle metabolites	Malonic acid fumaric acid, citric acid, and l-malic acid	Soybean	Scavenges reactive oxygen species	[75]

Table 7.

Details of a few metabolites observed under salt stress.

8.3.4 Ionomics

The process of analyzing elemental compositions on a large scale is known as ionomics. This approach has widespread uses in the field of plant sciences like screening mutants for the targeted traits, forward and reverse genetic techniques, and understanding the mechanisms involved in elemental/ion uptake, mobilization, compartmentalization, and exclusion [10]. This high-throughput analysis provides valuable insights into the intricate processes related to plant nutrition and ion homeostasis [76]. It will also help in understanding how cells function and adapt in response to abiotic stress [77]. Various analytical tools, including X-ray crystallography, ICP-MS, neutron activation analysis (NAA), and ICP-OES are utilized for the comprehensive profiling of ions in plants. To store and manage the vast amount of ionomic data, the purdue ionomics information management system (PiiMS) serves as a dedicated database. It serves as a valuable resource for storing and accessing the ionic profiles of plants [62]. During salinity stress, there will be excessive variation in the ionic composition. In that case, this technique helps in understanding how plants deal with salt stress by studying their ion profiles, which reveal how they detoxify ions and maintain ion balance under high-salt conditions. This technique, although not widely used in plants, holds great potential for studying mechanisms behind salinity.

8.4 Transgenic strategy for enhancing salt tolerance

In recent times, marker-assisted backcrossing and quantitative trait loci analysis have emerged as prominent strategies to improve salt tolerance in legumes. Nevertheless, these techniques are not without their limitations, such as linkage drag, marker validation, and breeding incompatibility across different species. Consequently, alternative biotechnological approaches, such as the genetic engineering of crops through the incorporation of genes responsible for imparting tolerance to abiotic stresses, hold great promise in facilitating the integration of functional, structural, and comparative genomics [24]. Transgenics refers to the transfer of desirable genes from related or unrelated species to the target host for enhancing the expression of desired traits. This approach is being used extensively worldwide for improving abiotic stress tolerance in plants. Gene transfer through Agrobacterium tumefaciens helps in achieving a significant improvement in legume transgenic experiments. However, the success rate of obtaining transgenic lines remains relatively low in various instances [78]. Several investigations have been conducted on manipulating the biosynthetic pathways of osmoprotectants to increase the production of molecules that aid in scavenging reactive oxygen species (ROS), diminishing lipid peroxidation, and preserving the structure and function of proteins (Table 8) [88]. Engineering salt-tolerant crops can be enhanced by, (a) good knowledge of posttranslational changes in the expression pattern of genes and proteins that ensure plant development in the salinity stress, (b) overexpression of miRNA, (c) regulating the hormone homeostasis to evade the multiple phenotypic effects of a gene (pleiotropic effects), (d) utilizing plant synthetic biology techniques to ameliorate genetic engineering approaches [23].

Gene	Source	Target plant	Role	Promoter used	Reference
AtNHX1	Arabidopsis (Arabidopsis thaliana)	Mung bean (Vigna radiata)	Compartmentalization of Na^+ ions into the vacuole.	CaMV 35S	[79]
OsRuvB	Rice (Oryza sativa)	Pigeon pea; (Cajanus cajan)	Increase the chlorophyll content, relative water content, peroxidase, and catalase activity.	CaMV 35S	[80]
Glyoxalase I	Mustard (Brassica juncea)	Mung bean (Vigna mungo)	Belongs to metalloglutathione transferase superfamily, detoxifies the cytotoxic methylglyoxal to S-D-lactoylglutathione, and involved in oxidative stress metabolism.	CmYLCV	[81]
GsERD15B	Soybean (Glycine max)	Soybean (Glycine max)	Improves the expressivity of genes associated with proline content, ABA-signaling, dehydration response, catalase-peroxidase, and cation transport	—	[82]
AhALDH3H1	Ground nut (Arachis hypogaea)	Soybean (Glycine max)	Oxidates aldehydes to protect cells from the aldehydes.	AhALDH3H1 promoter	[83]
VrNHX1	Mung bean (Vigna radiata)	Cowpea (Vigna unguiculata)	Sequestrates Na^+ from cytosol for compartmentalization in the vacuoles and maintains cellular homeostasis	CaMV 35S	[84]
OsDREB2A	Rice (Oryza sativa)	Soybean (Glycine max)	Accumulates osmolytes, improves the expressivity of a few key stress-inducible TFs and genes	—	[85]
P5CSF129A	Moth bean (Vigna aconitifolia)	Pigeon pea (Cajanus cajan)	Protects the cellular tissues through enhanced proline level. Increased proline reduces the level of free radicals.	CaMV 35S	[86]
GsCBRLK	Glycine soja	Alfalfa (Medicago sativa L.)	Enhancing superoxide dismutase (SOD) activity	CaMV 35S	[87]

Table 8.

Transgenic techniques employed in various legumes to enhance salinity tolerance.

9. Future directions and conclusion

Salinity is the most devastating abiotic stress ultimately affecting legume productivity. Several mechanisms like osmotic adjustment, ion homeostasis, ion exclusion, and compartmentalization are adapted by legumes to thrive under salinity stress. The prime concern nowadays is to develop cultivars that can alleviate/tolerate the ill effects of salinity and increase productivity. The knowledge of salt tolerance breeding is well-documented in cereals but not in legumes. With the advent of various molecular and omics techniques several markers, QTLs, and genes linked with saline stress tolerance have been identified which help improve the crop performance and productivity under saline conditions. Wild relatives possess numerous tolerance genes but due to the complex nature of the salt tolerance trait, understanding their roles at various developmental stages is required. Identification of a wild/cultivable donor requires the development or designing of appropriate screening procedures. Breeding for saline tolerance requires multi-environment screening which enables identification of tolerance-associated genomic regions. Utilization of appropriate molecular tools will improve the success rate. The database with integrated information on transcriptomics, metabolomics, and proteomics, especially for legumes, will help researchers understand the linkage between various signaling pathways like stress-specific, hormonal, and growth and developmental pathways. Recent advanced strategies like genomics selection (involving various mathematical models) help to predict the yield under abiotic stress conditions. Speed breeding is another approach that enables the growing of more crops in a year in a controlled condition (improves the breeding efficiency). Integrating these breeding approaches with other techniques like genomic, transcriptomics, metabolomics, transgenics as well as genetic engineering approaches like CRISPR-Cas9 are essential to develop legume varieties that can thrive in saline conditions and exhibit high-salt tolerance.

Acknowledgments

We acknowledge Dr. R. Ravikesavan, Director, Centre for Plant Breeding and Genetics, Dr. S. Geetha, Professor (PBG), and Head, Department of Pulses, for their technical support and critical suggestions.

Conflict of interest

None.