Salinity Tolerance Mechanisms in Rice: A Review

Rice is one of the world’s most important cereal crop which is grown over 156 mha area globally and is staple food crop of more than 50 per cent population worldwide (Fao, 2008). To maintain food security, rice production must be increased quantitatively and qualitatively to meet the requirements of the growing population in the twenty-first century (Rejeth et al., 2020). Although rice is adapted to a wide range of ecosystems it is susceptible to climatic changes leading to low rice productivity (Beena, 2005). Changing climate leads to periods of drought, frequent floods, sea water inundations, etc. which reduce the yield potential of current rice varieties. Among abiotic stresses, salinity is the second most devastating constraint in rice production after drought, affecting approximately 1 billion ha of land globally. Soil salinity affects about 800 million hectares of arable lands worldwide (Munns and Tester, 2008). Excessive use of irrigation water with improper drainage, poor quality irrigation water containing an excess level of salts, and flooding from seawater are the causes of salinity. Direct accumulation of salts disturbs metabolic processes and all major morpho-physiological and yield-related traits including tiller number, panicle length, spikelet number per panicle (Khatun et al., 1995), grain filling (Rao et al., 2013), plant biomass (Zeng et al., 2007) and photosynthesis (Baker 2008), leading to significantly decreased yield (Flowers and Yeo, 1981).

Salinity stress triggers a wide variety of plant responses, ranging from morphological, physiological, biochemical and molecular changes to plant system. Salt stress is first perceived by the root system and impairs plant growth both in the short term, by inducing osmotic stress caused by reduced water availability, and in the long term, by salt-induced ion toxicity due to nutrient imbalance in the cytosol. Therefore, the two main threats imposed by salinity are induced by osmotic stress and ionic toxicity associated with excessive Cl^- and Na⁺ uptake, leading to Ca²⁺ and K⁺ deficiency and to other nutrient imbalances (Marschner, 1995). In addition, salt stress is also manifested as oxidative stress mediated by ROS. All these responses to salinity contribute to the deleterious effects on plants. However, tolerance can come with trade-offs; for example, it has long been known that stress-tolerant plants have reduced growth rates and productivity (Beena et al., 2018c; Nithya et al., 2021). Hence, understanding the basis of tolerance is also important to understand the trade-offs between tolerance and growth/productivity for potent crop improvement.
 
Impacts of salinity stress on rice growth and development
 
Salt stress severely influences the morphology of rice plants which comprise of stunted plant growth, chlorosis, leaf burning, leaf rolling, poor root growth, wilting and senescence (Zhang et al., 2012). Salinity bring down the photosynthetic rate and associated parameters such as transpiration, stomatal conductance and intercellular CO₂‚ concentration (Munns, 2002). Salinity affects the pollination, fertilization and causes spikelet sterility in rice (Pearson and Bernstein,1959); delays heading and affect yield components (Grattan et al., 2002).

The biochemical impacts typically comprise of oxidative stress and ionic stress. Salt stress increases ROS production which can elevate oxygen–induced cellular damage. With the rise in salinity level, indices of oxidative stress viz., superoxide radical and H₂O₂‚ content increased in leaves of all the cultivars and this increase was more notable in salt-sensitive varieties and non-significant in resistant cultivars. There was 10 fold increase in Na⁺ concentration in salt sensitive “IR29” compared to salt tolerant rice variety “Pokkali” (Golldack et al., 2003). Elevated concentration of cytoplasmic Na⁺ and reduction of K⁺ result in changes of membrane potential, osmotic pressure, turgor pressure, calcium signaling, reactive oxygen species signaling, transcriptional regulation, alteration of gene expression, modification of protein expression pattern and spectra of siRNAs, signaling molecules, phytohormones and metabolites. NaCl at a concentration as low as 50 mM is lethal at the seedling stage (Yeo et al., 1990). High sodium concentrations over 100 mM often have inhibiting effect on protein synthesis at least in salt-sensitive glycophytes like rice (Flowers and Yeo, 1981).

Na+ is the principal poisonous ion in salinized soil and low Na⁺ /K⁺  ratio in the cytoplasm is essential to maintain a number of enzymatic reactions. Plants under salt stress normally absorb Na+ and simultaneously inhibit K⁺  absorption. High salt domains can shatter the ion homeostasis of plant cells, knock down the ionic balance and affect the distributions of ion at whole plant levels under salinity, increased osmotic pressure of the soil solution which restricts the water absorption and entry of water into the seeds, interruption of nutrient uptake, induces stomatal closure (Almansouri et al., 2001).

Salinity alters a wide array of metabolic processes in growing plants and induces changes in contents and activities of many enzymes. Sugar metabolism is critically affected when the plant grown under salinity condition. Under salinity, the starch content in roots declined more in salt sensitive cvs. Ratna and Jaya than in salt tolerant cvs. CSR-1 and CSR-3. The content of reducing and non-reducing sugars, and the activity of sucrose phosphate synthase was increased more in the sensitive than in the tolerant cultivars. Acid invertase activity decreased in shoots of the salt tolerant cultivars, whereas increased in salt sensitive cultivars. Starch phosphorylase activity decreased in all cultivars (Dubey and Singh, 1999). To better understand the impacts of salinity on plant growth, it is necessary to study the changes in metabolites involved in respiratory metabolism. A considerable reduction in saccharide abundance, including glucose, fructose, xylose, and sucrose has been reported in rice exposed to salinity (Sanchez et al., 2008). These saccharides are required as sources of glucose for glycolysis and may also contribute to osmotic adjustment in plants.
 
Mechanisms for salinity tolerance in rice
 
It is essential to know the basic molecular mechanisms of salt tolerance for the crop improvement programs to develop high yielding salt tolerant varieties. Salt tolerance is regarded as a complex quantitative trait which is controlled by multiple genes. Many adaptive responses at molecular, cellular and physiological level results in tolerance or resistance of rice plant to salt stress. There are mainly three mechanisms observed in rice.
 
Ion exclusion
 
Selective uptake of salt by root cells is the initial strategy followed by rice plant to tackle the salinity stress. Rahman et al., (2001) reported that root cap cell expansion is an adaptive response to salinity in rice plants and this root cap cells may function in Na⁺ exclusion under salinity stress. Once water is taken up by the root hair, it travels from cell to cell through intracellular regions across the cortex to handover the salt in xylem through apoplastic pathway. Suberin lamellae and casparian strip limit and force both water and salts to cross the plasma membrane into the cytoplasm, where they can continue on the symplast pathway, leading to decrease in Na+ accumulation in the shoot (Cai et al., 2011). With increase in NaCl concentration in rhizosphere, there will be a fall off of transpiration rate which lowers water potential in roots, and the transport of ABA from root to shoot and give rise to stomatal closure and prevent dehydration (Zhang et al., 2006).
 
Osmotic stress tolerance
 
Osmotic tolerance is the plant’s ability to tolerate the physiological drought aspect of salinity stress and to maintain leaf expansion and stomatal conductance by maintaining the tissue turgor (Rajendran et al., 2009). The early symptom of salinity stress on plants is osmotic stress which arise due to the accumulation of salts in the rhizosphere that affects negatively the plant growth (Munns and Tester, 2008). Compatible solutes accumulate in the cytosol, contribute to the decrease of cytoplasmic water potential and act as osmoprotectants. Rice plant under osmotic stress accumulates more carbohydrates or trehalose than proline (Nounjan and Theerakulpisut, 2012). Trehalose bring down Na⁺  accumulation, growth inhibition and salt (an osmotically regulated gene) expression at low to medium concentration and at higher concentration hinder NaCl^- induced loss of chlorophyll in blades, enhance rice growth and preserve root integrity (Garcia et al., 2012). Karthikeyan et al., (2011) observed high accumulation of proline in salt tolerant transgenic rice plants with expression of P5CS. codA introduced into transgenic rice plants promotes the synthesis of glycine betaine and enhancement of salt tolerance in plants (Su et al., 2006). Li et al., (2011) demonstrated that overexpression of OsTPS1 in rice plants enhances salt tolerance by increasing the amount of trehalose and proline.
 
Tissue tolerance
 
Tissue tolerance is achieved by sequestration of Na⁺ in the vacuole, synthesis of compatible solutes, production of enzymes catalyzing detoxification of reactive oxygen species. Reactive oxygen species (ROS) are dual functioning molecules which act both as toxic compounds as well as signal transduction molecules in response to abiotic as well as biotic stresses in plants (Miller et al., 2010). Under stress condition, there will be accumulation of plenty of ROS which will damage protein structure cause lipid peroxidation and mutation of DNA (Vaidyanathan et al., 2003). The antioxidant enzymes catalase (CAT) and ascorbate peroxidase (APX) are efficient scavengers of hydrogen peroxide (H₂O₂), and thus inhibit membrane lipid peroxidation, which is induced by high concentrations of H₂O₂. This is a key factor in reducing the effects of salinity stress (Mittler, 2007). Under saline condition, the capacity to scavenge reactive oxygen species decreased with age, and thus the apical region of the leaf blade suffered severe damage by Na⁺ than the basal region in salt sensitive rice variety (Yamane et al., 2009). In salinity tolerant rice cultivar, gene expression and the activity of antioxidant enzymes (SOD, CAT, APX and GR) were increased and protected from oxidative stress. Under salinity stress, salt tolerant rice cultivar Pokkali was reported to have higher activity of ROS scavenging enzymes such as catalase (CAT), ascorbate (ASC) and glutathione (GSH), compared to the salt sensitive rice cultivar (Pusa Basmati). In rice during salinity stress, cell death advances in a well-regulated manner and causes PCD in the root tip of rice (Liu et al., 2007). Transgenic rice plants containting antioxidative genes OsECS, OsVTE1 and OsMSRA4.1 showed high salt tolerance (Choe et al 2013).
 
Role of membrane transporters for salt tolerance in rice
 
Transporting Na+ out of the cytoplasm into apoplast or vacuole, which is driven, respectively, by Na⁺/H⁺ antiporters in plasma membrane or tonoplast, and exclusion of Na+ from sensitive shoot tissues are the basic strategies for the salt stressed plants to alleviate the Na⁺ toxicity (Hamamoto et al., 2015). Transporters could be considered as enzymes where conformational changes of a protein molecule are required for a complete transport cycle of ions (Gadsby, 2009). 

Turnover rate of the transporter is the number of complete transport cycles performed per second. There are three main types of transporters involved in salt tolerance mechanism of rice; Na+ transporters, such as vacuolar Na⁺/H⁺ antiporters (NHXs), salt overly sensitive 1 transporter (SOS1) and high-affinity potassium transporters (HKTs).
 
Vacuolar NHX  transporters 
 
The coupled exchange of K⁺ or Na⁺ for H⁺ is mediated by a family of transporters known as NHXs (NHX1-6). The cytosolic Na⁺ is compartmentalize Na⁺ into the vacuoles by NHXs  (Bassil and Blumwald, 2014). The compartmentation of Na⁺ into vacuoles not only reduces the toxicity of Na⁺ in the cytosol but also contributes to osmotic regulation. First evidence of NHX transporter in tonoplast vesicles from rice roots was reported by Fukuda et al., (1998). The mechanism of compartmentalization are the pH gradient generated by proton-translocating H⁺ -ATPase and proton translocating inorganic pyrophosphatase (H⁺- PPase) on the tonoplast and that the amount of antiporter in vacuolar membranes. Later, Fukuda et al., (2004) investigated the function and intracellular localization of the product of the OsNHX1 cloned from rice. Their results indicate that the OsNHX1 gene encodes a vacuolar (Na⁺, K⁺)/H⁺ antiporter. In addition, overexpression of OsNHX1 improved the salt tolerance of transgenic rice cells and plants, suggesting that OsNHX1 on the tonoplast plays important roles in removing accumulated Na⁺ and K⁺ in the cytoplasm into the vacuoles. Up to now, four vacuolar NHXs (OsNHX1-4) have been reported in rice (Kumar et al., 2013).
 
SOS transporter
 
SOS1 transporter was first isolated from rice (Martinez-Atienze et al., 2007). Based on genetic and biochemical criteria, they identified that OsSOS1 is a rice plasma membrane Na⁺/H⁺ antiporter and is functionally equivalent to the Arabidopsis SOS1. H⁺-ATPase present in plasma membrane is responsible for Na⁺ efflux from cytoplasm. The energy released from the hydrolysis of ATP is used to pump H⁺ out of the cell, generating electrochemical H+ gradient. The proton-motive-force generated by the H⁺-ATPase operates the Na⁺/H⁺ antiporters, which couple the movement of H⁺ into the cell along its electrochemical gradient to the efflux of Na⁺ against its electrochemical gradient.

In salt overly sensitive (SOS) pathway, salt stress elicited Ca²+ signals are perceived by Ca²⁺  binding protein SOS3 and activates SOS2, a serine/ threonine protein kinase (Halfter et al., 2000). Then the SOS2-SOS3 complex phosphorylates SOS1 which is a Na⁺ /H⁺ antiporter on cell membrane that extrudes Na+  out of the cytosol (Guo et al., 2004). The transport level of SOS1 is controlled by SOS₃-SOS₂ kinase complex. When there is a higher accumulation of Na⁺ in cytosol, SOS2 activates the tonoplast Na+/H+  antiporter  that sequesters Na+  into the vacuole (Blumwald, 2000). Elevation of salinity influences the Na⁺ /H⁺  antiporter activity and the activity is more significant in salt tolerant species than in salt sensitive ones.
 
HKT1 function in ion transport:
 
Sodium concentration in shoots is affected by the regulation of sodium transport from root to shoot by the family of HIGH-AFFINITY POTASSIUM TRANSPORTERS (HKTs). The members of this family can be divided into two classes according to their affinity for either sodium or potassium, caused by a single amino acid substitution in the pore region of the transporter. The HKT1 subclass transports sodium, while the HKT2 subclass transports potassium. OsHKT2; 2 is present in the salt-tolerant ‘Pokkali’ cultivar, but absent in ‘Nipponare’, can mediate both Na⁺ and K⁺ influx. This would explain why ‘Pokkali’ cutivar is salt tolerant (Garciadeblas et al., 2003). Identified three hybrid HKT gene (named OsHKT2;2/1; OsHKT2;1/2) in ‘Nona Bokra’ and OsHKT2;2  from ‘Pokkali’ (Oomen et al., 2012). These genes may contribute to the salt tolerance by maintaining K+ uptake under salinity conditions.
 
HAK/KUP family transporters
 
Importance of K⁺ retention ability in root and leaf tissues, role of HAK/KUP family of transporters in K⁺ uptake and its possible linkage with Ca²⁺ and ROS signaling, control of xylem K⁺ loading control of phloem K+ recirculation in salt stressed plants (Wu et al., 2018).
 
Role of ion channel proteins for salt tolerance in rice
 
Ion channels in an open state/conformation allow passage of over 106-108 ions per second via a selective pore formed within a protein molecule. The diameter of the pore is determined by the molecular structure of ion channel. The diameter of the pore and amino acids lining it essentially determine the ion selectivity of ion channel and potential number of passing ions per unit of time. Some of the important channel proteins present in rice are: OsAKT1 (K⁺ inwardrectifying channel), OsKCO1 (K⁺ outward-rectifying channel), OsTPC1 (Ca²⁺ permeable channel), OsCLC1 (Cl^– channel) and OsNRT1;2 (nitrate transporter).
 
Role of protein kinases for salt tolerance in rice
 
Important protein kinases involved in salt tolerance mechanism in rice are Mitogen-activated protein kinases (MAPKs), sucrose non-fermenting1-related kinase 2 (SnRK2) family members have been designated as stressactivated protein kinases (OsSAPK), receptor-like cytoplasmic kinases (RLCKs). Over-expression of OsMAPK5 (encoding an abscisic acid-inducible MAPK), enhances salt tolerance in rice (Xiong and Yang, 2003). OsMAPK44, reduced the salt injury by maintaining ion homeostasis in transgenic rice (Jeong et al., 2006). OsMAPK33 exhibits salinity tolerance in transgenic rice by altered expression of ion transport genes such as the K⁺ /H⁺ antiporter or other downstream positive regulators of salt tolerance in the MAPK pathway (Lee et al., 2011). The expressions of all the ten OsSAPKs (stress activated protein kinases) are induced under salt stress conditions by osmotic stress and/or ABA dependent signaling (Cutler et al., 2010). Overexpression of SAPK4 improved germination, growth and development in seedlings and mature rice plants under salinity following alteration in the expression of genes involved in ion homeostasis and oxidative stress responses (Diédhiou et al., 2008). Overexpression of SAPK6 in rice up-regulates the expression of the enzymes involved in diverse salt stress-responsive metabolic pathways under salt stress conditions.
 
Role of transcription factors and miRNAs for salt tolerance in rice
 
Transcription factors (TFs) are protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. Several salt responsive TFs were identified in rice, OsCOIN (Liu et al., 2007), OsNAC6 (Nakashima et al., 2007), OsNAC5 (Takasaki et al., 2010), OsDREB2A (Mallikarjuna et al., 2011), OsMYB2 (Yang et al., 2012), OsTZF1 (Jan et al., 2013), OsBZIP71 (Liu et al., 2014) which improve salt tolerance by various mechanisms like the accumulation of osmoprotectants and antioxidants, increase transporter activity for Na⁺ and K⁺ ions, regulate the expression of other saltresponsive genes.

By modulating the gene expression at the post-transcriptional level, miRNAs can regulate plant development and respond to environmental stress (Table 1).


 
Approaches to improve salt tolerance in rice
 
Conventional breeding methods such as use of in vitro selection, interspecific hybridization and pooling physiological etc. were used to enhance salinity tolerance (Flowers, 2004). Superior structured design to breeding for salinity tolerance acquired when there was a better understanding of the function regulation and function of the responsive genes and their association to QTL regions (Negrao et al., 2012). Fig 1 shows the different approaches used for developing salt tolerant rice varieties. In India, there were many success stories in developing and releasing the salt tolerant cultivars including CSR10, CSR13, CSR27, Narendra usar 2 and Narendra usar 3 (Singh et al., 2004) and those varieties have been used to reclaim saline soils in India.



CSR 2, CSR 3, CSR 13, CSR 22, CSR 23, CSR 26, CSR 27, CSR 30, Panvel 1, Panvel 2, Panvel 3, Vyttila 1 and Vyttila 2 are the varieties developed for the coastal saline soil conventionally (Shahbaz and Ashraf, 2013). Kerala Agricultural University has released 11 Vytilla series of rice verieties.

A major QTL saltol was identified by Gregorio and Senadhira in salinity tolerant Pokkali responsible for maintaining Na⁺/K⁺ homeostasis at the seedling stage (Gregorio et al., 2002).  Marker-assisted backcross breeding (MABB) was attempted to incorporate the Saltol loci from Pokkali or its derivative FL478 facilitate the development of salinity-tolerant rice varieties (Valarmathi et al., 2019). Saltol gene introgressed Pusa Basmati 1 (PB1) showed enhanced seeding stage salt tolerance (Singh et al., 2018). Under salinity stress, saltol introgressed backcross lines showed salt tolerance by maintaining good morphological and yield traits (Banumathy et al., 2018).

A salt-tolerant rice mutant rst1 was isolated and characterized which showed significantly lower lipid peroxidation and electrolyte leakage under salinity stress but higher shoot biomass and chlorophyll content (Deng et al., 2015). Microarray analysis of rice mutant RGA1 disclosed its role in regulation of multiple abiotic stresses (Jangam et al., 2016). One mutant variety (Shua-92) and two mutants (IR8-202 and Pokkali-M) were developed from rice varieties IR8 and Pokkali through mutation breeding, evaluated to check the yield performance for two years under salinity stress and results showed that the mutant variety Shua-92 produced 40-49 per cent more paddy yield on saline soil than the famous salinity tolerant Nona Bokra and Pokkali varieties (Baloch et al., 2003).

Exploiting the potential of beneficial microorganisms is an alternative strategy for ameliorating plant stress tolerance. Stress alleviation is ensured by signaling events occurring during the plant–microbe interaction and these mechanisms are regulated by a complex network of signaling events (Smith et al., 2017). Recent studies showed that plant growth promoting bacteria act as growth promoter as well as elicitors of salinity tolerance (Tiwari et al., 2016).

There are many recent developments in the understanding of the mechanisms of salt tolerance such as RNA guided CRISPR endonuclease technology including base and prime editing or targeted mutagenesis which help in gene discovery, functional analysis transferring favorable alleles into elite breeding line to accelerate the breeding of salt-tolerant rice cultivars (Ganie, 2021). As a future line of study, the advanced technologies such as, genome editing can be used as an excellent tool, for improving crop traits, with salinity stress tolerance, as a target.

Na⁺ exclusion from the root, modulation of root–shoot transport, and cellular compartmentalization of Na⁺, as well as maintenance of cytoplasmic osmotic balance, are crucial aspects of salt tolerance. HKT1 plays an important role in root stellar cells, modulating sodium transport to the shoot. Tissue-specific expression of HKT1 at specific developmental stages can contribute to plant salt tolerance. By targeting and phosphorylating downstream components, both ABA-independent SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE2 (SnRK2s) and ABA-dependent SnRK2s play pivotal roles in transcriptional regulation and posttranscriptional regulation during salt stress response.