Physiology of Salt Tolerance

Ashwani Kumar, Anshuman Singh, Jogendra Singh, Pooja and Vijayata Singh Central Soil Salinity Research Institute, Karnal-132004, Haryana (India) World agriculture is facing a lot of challenges like producing 70% more food for an additional 2.3 billion people by 2050. Over the last few decades, achieving sustainability in agriculture has emerged as a major goal to fulfill the requirements of enough food to feed a rapidly increasing world population in changing environmental conditions. Agricultural productivity is highly influenced by abiotic stresses, are the primary cause of crop failure, causing average yield losses of more than 60% for major crops worldwide (Bray et al., 2000). Several environmental factors adversely affect plant growth and development and final yield performance of a crop. Drought, salinity, nutrient imbalances (including mineral toxicities and deficiencies) and extremes of temperature are among the major environmental constraints to crop productivity worldwide.

Figure 1: Plant responses to environmental stress in correspondence with stress and plant characteristics. Salinity is one of the most serious factors limiting the productivity of agricultural crops, with adverse effects on germination, plant vigour and crop yield (Munns & Tester, 2008). Today, ~20% of the worlds cultivated land and nearly half of all irrigated lands are affected by salinity

(Yeo, 1999). High concentrations of salts causes ion imbalance and hyperosmotic stress in plants. As a consequence of these primary effects, secondary stresses such as oxidative damage often occur. High salinity affects plants in several ways: water stress, ion toxicity, nutritional disorders, oxidative stress, alteration of metabolic processes, membrane disorganization, reduction of cell division and expansion, genotoxicity (Munns, 2002; Zhu, 2007). Together, these effects reduce plant growth, development and survival. Fundamentally, plants cope by either avoiding or tolerating salt stress. That is plants are either dormant during the salt episode or there must be cellular adjust to tolerate the saline environment. Based on their capacity to grow on high salt medium, plants are traditionally classified as glycophytes or halophytes (Flowers et al., 1977). Halophytes are tolerant to high concentrations of NaCl; some can withstand salts that are more than twice the concentration of seawater. Most plants, including the majority of crop species, are glycophytes and cannot tolerate high salinity. For glycophytes, salinity imposes ionic stress, osmotic stress, and secondary stresses such as nutritional disorders and oxidative stress. Sodium toxicity represents the major ionic stress associated with high salinity. In certain saline soils, ion toxicity is worsened by alkaline pH. The low osmotic potential of saline solutions hampers plant water uptake, resulting in physiological drought.

Fig. 2. Two-phase growth response to salinity (Adapted from Munns 1995).

Causes and Tolerance of Salinity Chapman (1966), saline soil owes its origin in single or combination of the following factors as a primary or secondary type. Primary salinity arises due to 1) weathering of rocks, 2) capillary rise from shallow brackish groundwater, 3) intrusion of seawater along the coast, 4) salt laden sand blown by sea winds and 5) impeded drainage. While, secondary salinization is the result of human activities such as introduction of irrigation without proper drainage system, industrial effluents, overuse of fertilizers, removal of natural plant cover and flooding with salt rich waters, high water table and the use of poor quality groundwater for irrigation.

Fig 3: Salinity and its detrimental effects (Adapted from Horie et al. 2012). Salt stress causes reduction in plant growth because plant may suffer four types of stresses (1) Osmotically induced water stress, (2) Specific ion toxicity due to high concentration of sodium and chloride, (3) Nutrient ion imbalance, due to high level of Na+ and Cl- which reduce the uptake of

K+, NO-, PO43- etc., (4) Increased production of reactive oxygen species which damage the macromolecules. (Greenway and Munns, 1980) Tolerance mechanisms can be categorized as those that function to minimize osmotic stress or ion disequilibrium or alleviate the consequent secondary effects caused by these stresses. The chemical potential of the saline solution initially establishes a water potential imbalance between the apoplast and symplast that leads to turgor decrease, which if severe enough can cause growth reduction (Bohnert et al., 1995). Growth cessation occurs when turgor is reduced below the yield threshold of the cell wall. Cellular dehydration begins when the water potential difference is greater than can be compensated for by turgor loss (Taiz and Zeiger, 1998). A) Challenges to Salt stress tolerance

Plants use three main mechanisms with which to tolerate salinity stress (Munns and Tester, 2008): 1) Osmotic-stress tolerance, the ability to maintain growth under osmotic stress, a process that causes stomatal closure and reduced cell expansion in root tips and leaves, 2) Na+ exclusion, the reduction of Na+ accumulation in shoots by Na+ exclusion in the roots, 3) Tolerance of tissues to accumulated Na+ and possibly Cl, requiring, in most cases, compartmentation of Na+ and Cl at the cellular and intracellular levels.

MECHANISMS OF SALT TOLERANCE 1. Osmotic Adjustment/Tolerance

The cellular response to turgor reduction is osmotic adjustment. The osmotic adjustment is achieved in these compartments by accumulation of compatible osmolytes and osmoprotectants (Bohnert, 1995; Bohnert and Jensen, 1996). Since plant cell growth occurs primarily because of directional expansion mediated by an increase in vacuolar volume, compartmentalization of Na+ and Cl- facilitates osmotic adjustment that is essential for cellular

development. Salt stress reduces the plants ability to take up water, and this leads to reduction in growth. This is the osmotic or water-deficit effect of salt stress. Osmotic tolerance involves the plants ability to tolerate the drought aspect of salinity stress and to maintain leaf expansion and stomatal conductance (Rajendran, Tester & Roy, 2009). Both cellular and metabolic processes involved in osmotic stress due to salinity are common to drought. Osmolytes are synthesized as a metabolic response to salt stress, and include sugars, polyols, amino acids, and tertiary and quaternary ammonium and sulphonium compounds (Chen and Murata 2002). Others include quaternary amino acid derivatives (proline, glycine betaine, alanine betaine, proline betaine), tertiary amines 1, 4, 5, 6-tetrahydro-2-mehyl-4-carboxyl pyrimidine), and sulfonium compounds (choline-o-sulfate, dimethyl sulfonium propironate) (Nuccio et al., 1999). Many organic osmolytes are presumed to be osmoprotectants, as their levels of accumulation are insufficient to facilitate osmotic adjustment. Compatible/Osmotic solutes are polar, highly soluble and typically hydrophilic so that their protective function might be in maintaining the hydration sphere of proteins without interfering with normal metabolism and accumulate predominantly in the cytoplasm at high concentrations under osmotic stress. High concentrations of these substances protect proteins from misfolding and hence act as low molecular weight chaperones, stabilize some macromolecules or molecular assemblies, reduce the inhibitory effects of ions on enzyme activity to increase their thermal stability, and prevent dissociation of enzyme complexes. They alleviate the toxic effect of reactive oxygen species (ROS) generated by salt stress, act as scavengers of hydroxyl radicals (extremely toxic, short lived active oxygen species), thus preserving either enzyme activity or membrane integrity. An adaptive biochemical function of osmoprotectants is the scavenging of reactive oxygen species that are byproducts of hyperosmotic and ionic stresses and cause membrane dysfunction and cell death (Bohnert and Jensen, 1996).

2.

Ion Homeostasis During Salt Stress/Specific Ion Toxicity

Na+ Toxicity and Homeostasis The process of ion accumulation for osmotic adjustment, some ions can leak into the transpiration stream (Yeo et al. 1987) and that such leaks must be minimized if the aerial parts are not to be swamped by ions (Munns 2005).

Fig. 4: Possible mechanisms of plants to adjust at high external NaCl salinity [ Modified after Marschner, 1995]. Similar to osmotic stress, high concentrations of Na+ in the soil/increased Na+ accumulation in the plant system may be recognized by extracellular and intracellular sensors such that the effective counteracting mechanisms will be initiated. Salinity causes ion-specific stresses resulting from altered K+/Na+ ratios leads to build up in Na+ and Cl concentrations that are detrimental to plants. The alteration of ion ratios in plants is due to the influx of Na+ through pathways that function in the acquisition of K+. Maintenance of a high cytosolic K+/Na+ ratio is a key requirement for plant growth under high concentration of salt (Yamaguchi and Blumwald, 2005). Accumulation of sodium in the cytoplasm is prevented by restricting its uptake across the plasma membrane and by promoting its extrusion or sequestration in halophytes (Hasegawa et al., 2000). Ion transporters are considered to play an important role in salt tolerance. In principle, three mechanisms exist to prevent excess Na+ accumulation in the symplast of plant cells: 1. Restricting the Na+ permeation and entry into plants by Na+ transporters, whose molecular identity is unknown.

2. Compartmentalizing the Na+ in the vacuole. 3. Extruding Na+: cytosolic Na+ can be transported back to the external medium or the apoplast via plasma membrane Na+/H+ antiporter activity. Mechanisms for salt tolerance are therefore of two main types: those minimizing the entry of salt into the plant, and those minimizing the concentration of salt in the cytoplasm. Low salt transport to leaves the mechanism known as salt exclusion Salt exclusion functions to reduce the rate at which salt accumulates in transpiring organs. Salts carried in the transpiration stream are deposited in leaves as the water evaporates, and salt gradually builds up with time. The salt concentrations in older leaves are therefore much higher than in younger leaves, at any one point in time. The mechanisms by which salt is excluded from leaves are: 1. Selectivity of uptake by root cells. It is still unclear which cell types control the selectivity of ions from the soil solution. The initial uptake of Na+ and Cl could occur at the epidermis, at the exodermis, or if soil solution flows apoplastically across the root cortex, it would occur at the endodermis. 2 Loading of the xylem. There is evidence for a preferential loading of K+ rather than Na+ by the stellar cells that is under genetic control (Gorham et al., 1990). 3 Removal of salt from the xylem in the upper part of the roots, the stem, petiole or leaf sheaths. In many species, Na+ is retained in the upper part of the root system and in the lower part of the shoot, indicating an exchange of K+ for Na+ by the cells lining the transpiration stream.

3.

Reactive Oxygen Species and Antioxidants: Dual functions in plant stress response
Chloroplasts, mitochondria and peroxisomes are the most important intracellular

generators of ROS like O2. and H2O2. Amongst these, chloroplast-mediated O2. and H2O2 production remains the most unavoidable consequence of an oxygen-enriched atmosphere. In

chloroplasts, O2. and H2O2 are mainly produced by the electron acceptor of photosystem I, whereas singlet oxygen is generated by the transfer of an electron from an excited chlorophyll molecule to molecular oxygen (Asada and Takahashi, 1987; Hernandez et al. 1995). ROS are also observed in mitochondrial electron transport (Zhang et al. 1990; Hernandez et al. 1993; Hamilton and Heckathorn, 2001). ROS are predominantly generated in the chloroplast by direct transfer of excitation energy from chlorophyll to produce singlet oxygen, or by univalent oxygen reduction at photosystem I, in the Mehlers reaction (Foyer et al., 1994; Allen, 1995) and to some extent in mitochondria. Chloroplasts are the first targets in plant cells since this is the major site of ROS production. The increased concentration of ROS inhibits the ability to repair damage to photosystem II and inhibits the synthesis of the D1 protein. Stress-enhanced photorespiration and NADPH activity also contributes to the increased H2O2 accumulation, which may inactivate enzymes by oxidizing their thiol groups. ROS have the potential to cause oxidative damage to cells during environmental stress. The active oxygen species such as superoxide (O2), hydrogen peroxide (H2O2), hydroxyl radical (OH), and singlet oxygen (1O2) are produced during normal aerobic metabolism when electrons from the electron transport chains in mitochondria and chloroplasts are leaked and react with O2 in the absence of other acceptors [145,146]. However, plants generally have the ability to eliminate superoxide with the help of superoxide dismutase (SOD), which catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen, and is important in preventing the reduction of metal ions and hence the synthesis of hydroxyl radicals. Hydrogen peroxide can be eliminated by an ascorbate peroxidase located in the thylakoid membrane [147]. Earlier studies confirm that in plants subjected to NaCl stress, the balance between the production of ROS and the quenching activity of antioxidants is upset, resulting in oxidative stress damage (Hernandez et al. 1999). The active oxygen species are highly reactive and, in the absence of a protective mechanism in plants, can cause serious damage to different aspects of cell structure and function. Plants, therefore, are adequately protected by a number of anti-oxidative mechanisms to scavenge free radicals. The plant anti-oxidative stress pathway comprises two components, the non enzymatic and the enzymatic components. The non enzymatic component consists of antioxidants such as

tocopherol, carotenoids, ascorbate and glutathione that are free-radical-scavenging molecules (Salin 1987). The enzymatic component consists of enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, mono hydroascorbate reductase, dehydro ascorbate reductase and glutathione reductase (Salin 1987). Apart from these, an iron-storage protein, ferritin, is also involved in the reactive-oxygen scavenging network (Morel and Barouki, 1999; Mittler et al. 2004). In these cases, enhancement of antioxidant functions has been suggested to be the more radical approach for increasing stress tolerance capacity because (i) Water stress imposed by increased salinity disrupts cellular homeostasis and chloroplast functions (Bohnert and Jensen, 1996) and (ii) Antioxidants have been implicated in stress acclimation (Dat et al. 2000).

Engineering salt tolerance in plants

Plant adaptation to environmental stresses is controlled by cascades of molecular networks. Physiologic or metabolic adaptations to salt stress at the cellular level are the main responses amenable to molecular analysis and have led to the identification of a large number of genes induced by salt (Ingram and Bartels, 1996; Bray, 1997; Shinozaki et al., 1998). These activate stress responsive mechanisms to re-establish homeostasis and to protect and repair damaged proteins and membranes (Fig. 5). In contrast to plant resistance to biotic stresses, which is mostly dependent on monogenic traits, the genetically complex responses to abiotic stresses are multigenic and thus more difficult to control and engineer. Primary stresses, such as drought, salinity, cold, heat and chemical pollution, are often interconnected and cause cellular damage and secondary stresses, such as osmotic and oxidative stress. The initial stress signals (e.g. osmotic and ionic effects or changes in temperature or membrane fluidity) trigger the downstream signaling process and transcription controls, which activate stress-responsive mechanisms to re-establish homeostasis and to protect and repair damaged proteins and membranes. Inadequate responses at one or more steps in the signaling and gene activation process might ultimately result in irreversible changes in cellular homeostasis and in the destruction of functional and structural proteins and membranes, leading to cell death.

Fig. 5: The complexity of the plant response to abiotic stress Salt tolerance is a multigenic trait and a number of genes categorized into different functional groups are responsible for encoding salt-stress proteins: genes for photosynthetic enzymes, genes for synthesis of compatible solutes, genes for vacuolar-sequestering enzymes, and genes for radical-scavenging enzymes. Most of the genes in the functional groups have been identified as salt inducible under stress conditions.

Acquired plant stress tolerance can be enhanced by manipulating stress-associated genes and proteins and by over expression of stress associated metabolites. Plant resistance to abiotic stress is a multigenic trait, depending on the combination of many genes, proteins and metabolic pathways all playing in concert. Stress-associated mechanisms that are not discussed in the present review are marked by an asterisk. Acquired plant tolerance to abiotic stress can be achieved both by genetic engineering and by conventional plant breeding combined with the use of molecular markers and quantitative trait loci (QTLs). Hsp, heat shock protein; LEA, late embryogenesis abundant; ROS, reactive oxygen species. A more complete understanding of the complexity and interplay of osmotic, desiccation and temperature tolerance mechanisms, and their corresponding signaling pathways, is therefore needed and will come from integrative, whole genome studies (Bouchez and Hfte, 1998; Somerville and Somerville, 1999).

Conclusion
Plants develop a plethora of biochemical and molecular mechanisms to cope with salt stress. Biochemical pathways leading to products and processes that improve salt tolerance are likely to act additively and probably synergistically (Iyengar and Reddy, 1996).

Biochemical functions associated with tolerance to plant salt stress. (Reproduced from Bohnert and Jensen, 1996). References: 1. Bouchez D, Hfte H: Functional genomics in plants. Plant Physiol 1998, 118:725-732. 2. Somerville C, Somerville S: Plant functional genomics. Science 1999, 285:380-383. 3. Bohnert, H.J., Jensen, R.G., 1996. Strategies for engineering water stress tolerance in plants. Trends Biotechnol. 14, 8997.

4. Iyengar, E.R.R., Reddy, M.P., 1996. Photosynthesis in highly salt tolerant plants. In: Pesserkali, M. (Ed.), Handbook of photosynthesis. Marshal Dekar, Baten Rose, USA, pp. 897909. 5. Ingram, J., Bartels, D., 1996. The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 377403. 6. Bray, E.A., 1997. Plant responses to water deficit. Trends Plant Sci. 2, 4854. 7. Shinozaki, K., Yamaguchi-Shinozaki, K., Mizoguchi, T., Uraro, T., Katagiri, T., Nakashima, K., Abe, H., Ichimura, K., Liu, Q.A., Nanjyo, T., Uno, Y., Luchi, S., Srki, M., Lto, T., Hirayama, T., Mikami, K., 1998. Molecular responses to water stress in Arabidopsis thaliana. J. Plant Res. 111, 345351. 8. Asada, K., Takahashi, M., 1987. Production and scavenging of active oxygen radicals in photosynthesis. In: Kyle, D.J., Osmond, C.B., Arntzen, C.J. (Eds.), Photoinhibition, vol. 9. Elsivier, Amsterdam, pp. 227288. 9. Bohnert, H.J., Nelson, D.E., Jensen, R.G., 1995. Adaptations to environmental stresses. Plant Cell 7, 10991111. 10. Flowers, T.J., Troke, P.F., Yeo, A.R., 1977. The mechanism of salt tolerance in halophytes. Annu. Rev. Plant Physiol. 28, 89121. 11. Foyer, C.H., Lelandais, M., Kunert, K.J., 1994. Photooxidative stress in plants. Physiol. Plant. 92, 696717. 12. Greenway, H., Munns, R., 1980. Mechanisms of salt tolerance in nonhalophytes. Annu. Rev. Plant Physiol. 31, 149190. 13. Hernandez, J.A., Olmos, E., Corpas, F.J., Sevilla, F., del Rio, L.A., 1995. Salt-induced oxidative stress in chloroplasts of pea plants. Plant Sci. 105, 151167. 14. Hernandez, J.A., Campillo, A., Jimenez, A., Alacon, J.J., Sevilla, F., 1999. Response of antioxidant systems and leaf water relations to NaCl stress in pea plants. NewPhytol. 141, 241251. 15. Hasegawa, P.M., Bressan, R.A., Zhu, J.K., Bohnert, H.J., 2000. Plant cellular and molecular responses to high salinity. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 463 499. 16. Munns, R. (2002). Comparative physiology of salt and water stress. Plant Cell Environ. 25, 239-250.

17. Nuccio, M.L., Rhodes, D., McNeil, S.D. and Hanson, A.D. (1999) Metabolic engineering of plants for osmotic stress resistance. Curr. Opin. Plant Biol. 2, 128-34. 18. Rajendran, K., Tester, M., & Roy, S. J. (2009). Quantifying the three main components of salinity tolerance in cereals. Plant, Cell & Environment, 32(3), 237-249.