Salt tolerance and salinity effects on plants: a review

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Ecotoxicology and Environmental Safety 60 (2005) 324–349 www.elsevier.com/locate/ecoenv

Salt tolerance and salinity effects on plants: a review Asish Kumar Paridaa, Anath Bandhu Dasa,b, a

National Institute for Plant Biodiversity Conservation and Research, Nayapalli, Bhubaneswar 751015, Orissa, India b Regional Plant Resource Centre, Nayapalli, Bhubaneswar 751015, Orissa, India Received 18 September 2003; received in revised form 8 March 2004; accepted 8 June 2004 Available online 7 August 2004

Abstract Plants exposed to salt stress undergo changes in their environment. The ability of plants to tolerate salt is determined by multiple biochemical pathways that facilitate retention and/or acquisition of water, protect chloroplast functions, and maintain ion homeostasis. Essential pathways include those that lead to synthesis of osmotically active metabolites, specific proteins, and certain free radical scavenging enzymes that control ion and water flux and support scavenging of oxygen radicals or chaperones. The ability of plants to detoxify radicals under conditions of salt stress is probably the most critical requirement. Many salt-tolerant species accumulate methylated metabolites, which play crucial dual roles as osmoprotectants and as radical scavengers. Their synthesis is correlated with stress-induced enhancement of photorespiration. In this paper, plant responses to salinity stress are reviewed with emphasis on physiological, biochemical, and molecular mechanisms of salt tolerance. This review may help in interdisciplinary studies to assess the ecological significance of salt stress. r 2004 Elsevier Inc. All rights reserved. Keywords: Antioxidative enzymes; Compatible solutes; Ion homeostasis; Photosynthesis; Salt stress

1. Introduction Salinity is the major environmental factor limiting plant growth and productivity (Allakhverdiev et al., 2000b). The detrimental effects of high salinity on plants can be observed at the whole-plant level as the death of plants and/or decreases in productivity. Many plants develop mechanisms either to exclude salt from their cells or to tolerate its presence within the cells. During the onset and development of salt stress within a plant, all the major processes such as photosynthesis, protein synthesis, and energy and lipid metabolism are affected. The earliest response is a reduction in the rate of leaf surface expansion, followed by a cessation of expansion as the stress intensifies. Growth resumes when the stress is relieved. Carbohydrates, which among other subCorresponding author. National Institute for Plant Biodiversity Conservation and Research, Nayapalli, Bhubaneswar 751015, Orissa, India. Fax: +91-674-2550274. E-mail address: [email protected] (A.B. Das).

0147-6513/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.06.010

strates are needed for cell growth, are supplied mainly through the process of photosynthesis, and photosynthesis rates are usually lower in plants exposed to salinity and especially to NaCl. Salinity stress biology and plant responses to high salinity have been discussed over two decades (Flowers et al., 1977; Greenway and Munns, 1980; Ehret and Plant, 1999; Hasegawa et al., 2000; Zhu, 2002) and it has been over a decade since salinity tolerance in marine algae has been covered (Kirst, 1989). These reviews covered organismal, physiological, and the then-known biochemical hallmarks of stress and the bewildering complexity of plant stress responses. We summarize in this review physiological, biochemical, and molecular mechanisms of salt tolerance with the salient features of salinity stress effects on plants. In this review, much research information about cellular, metabolic, molecular, and genetic processes associated with the response to salt stress, some of which presumably function to mediate salt tolerance, has been gathered.

ARTICLE IN PRESS A.K. Parida, A.B. Das / Ecotoxicology and Environmental Safety 60 (2005) 324–349

2. Salt tolerance of plants Salt tolerance is the ability of plants to grow and complete their life cycle on a substrate that contains high concentrations of soluble salt. Plants that can survive on high concentrations of salt in the rhizosphere and grow well are called halophytes. Depending on their salt-tolerating capacity, halophytes are either obligate and characterized by low morphological and taxonomical diversity with relative growth rates increasing up to 50% sea water or facultative and found in less saline habitats along the border between saline and nonsaline upland and characterized by broader physiological diversity which enables them to cope with saline and nonsaline conditions. 2.1. Mechanism of salt tolerance 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 strategies include (i) selective accumulation or exclusion of ions, (ii) control of ion uptake by roots and transport into leaves, (iii) compartmentalization of ions at the cellular and whole-plant levels, (iv) synthesis

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of compatible solutes, (v) change in photosynthetic pathway, (vi) alteration in membrane structure, (vii) induction of antioxidative enzymes, and (viii) induction of plant hormones (Fig. 1). These are discussed under separate headings. Salt tolerance mechanisms are either low-complexity or high-complexity mechanisms. Low-complexity mechanisms appear to involve changes in many biochemical pathways. High-complexity mechanisms involve changes that protect major processes such as photosynthesis and respiration, e.g., water use efficiency, and those that preserve such important features as cytoskeleton, cell wall, or plasma membrane–cell wall interactions (Botella et al., 1994) and chromosome and chromatin structure changes, i.e., DNA methylation, polyploidization, amplification of specific sequences, or DNA elimination (Walbot and Cullis, 1985). It is believed that for the protection of higher-order processes, low-complexity mechanisms are induced coordinately (Bohnert et al., 1995). 2.2. Ion regulation and compartmentalization Ion uptake and compartmentalization are crucial not only for normal growth but also for growth under saline conditions (Adams et al., 1992b) because the stress disturbs ion homeostasis. Plants, whether glycophyte or

Extracellular and cell-wall space• Ion exclusion • Ion export • Cell-wall modification

Cytosol and organelle space • Osmotic adjustment • Radical scavenging • Ion-selectivity changes • Enhancement of proton pumping • Aquaporin-activity control

• Plant growth regulator sensitivity adjustment • Osmoprotection • Ion partitioning

Vacuolar space • Ion (sodium, calcium) storage • Ion (potassium) export • Osmotic adjustment • Proton-gradient maintenance

Fig. 1. Biochemical functions associated with tolerance to plant salt stress. The schematic presentation of a plant cell includes three compartments that are defined by the plasma membrane and tonoplast (reproduced from Bohnert and Jensen, 1996).

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A.K. Parida, A.B. Das / Ecotoxicology and Environmental Safety 60 (2005) 324–349

halophyte, cannot tolerate large amounts of salt in the cytoplasm and therefore under saline conditions they either restrict the excess salts in the vacuole or compartmentalize the ions in different tissues to facilitate their metabolic functions (Reddy et al., 1992; Iyengar and Reddy, 1996; Zhu, 2003). Glycophytes limit sodium uptake or partition sodium in older tissues that serve as storage compartments that are eventually sacrificed (Cheeseman, 1988). Removal of sodium from the cytoplasm or compartmentalization in the vacuoles is done by a salt-inducible enzyme Na+/H+ antiporter (Apse et al., 1999). Two electrogenic H+ pumps, the vacuolar type H+-ATPase (V-ATPase) and the vacuolar pyrophosphatase (VPPase), coexist at membranes of the secretory pathway of plants (Dietz et al., 2001). The V-ATPase is the dominant H+ pump at endomembranes of most plant cells with regard to protein amount and, frequently, activity. The V-ATPase is indispensable for plant growth under normal conditions due to its roles in energizing secondary transport, maintaining solute homeostasis, and, possibly, facilitating vesicle fusion. Under stress conditions such as salinity, drought, cold, acid stress, anoxia, and excess heavy metals in the soil, survival of the cells depends strongly on maintaining or adjusting the activity of the V-ATPase. Regulation of gene expression and activity are involved in adapting the V-ATPase on long- and short-term bases (Dietz, et al., 2001). Salt modulation of the tonoplast H+-pumping VATPase and H+-pyrophosphatase has been evaluated in hypocotyls of Vigna unguiculata seedlings (Otoch et al., 2001). Salt stress induces V-ATPase expression in V. unguiculata with concomitant enhancement of its activity as a homeostatic mechanism to cope with salt stress, whereas under the same conditions V-PPase is inhibited (Otoch et al., 2001). The main strategy of salt tolerance in the halophyte Suaeda salsa seems to be an up-regulation of V-ATPase activity, which is required to energize the tonoplast for ion uptake into the vacuole, while V-PPase plays only a minor role (Wang et al., 2001). When under salt stress, plants maintain high concentrations of K+ and low concentrations of Na+ in the cytosol. They do this by regulating the expression and activity of K+ and Na+ transporters and of H+ pumps that generate the driving force for transport (Zhu et al., 1993). Although salt-stress sensors remain elusive, some of the intermediary signaling components have been identified. Evidence suggests that a protein kinase complex consisting of the myristoylated calcium-binding protein SOS3 and the serine/threonine protein kinase SOS2 is activated by a salt-stress-elicited calcium signal. The protein kinase complex then phosphorylates and activates various ion transporters, such as the plasma membrane Na+/H+ antiporter SOS1 (Zhu et al., 1993).

The Arabidopsis thaliana AtNHX1 gene encodes a vacuolar Na+/H+ antiporter that is important in salt tolerance. The tissue distribution and regulation of AtNHX1 expression by salt stress and abscisic acid (ABA) have been reported by Shi and Zhu (2002). Experimental evidence shows that the steady state level of AtNHX1 transcript is up-regulated by treatment with NaCl, KCl, or ABA. AtNHX1 promoter (GUS) analysis in transgenic Arabidopsis shows that AtNHX1 is expressed in all tissues except the root tip. Strong GUS expression was detected in guard cells, suggesting that AtNHX1 may play a role in pH regulation and/or K+ homeostasis in the specialized cells. AtNHX1 promoter activity is substantially up-regulated by NaCl, KCl, or ABA, demonstrating that salt and ABA regulation of AtNHX1 expression occurs at the transcriptional level. Strong induction of GUS activity in root hair cells suggests a role of AtNHX1 in storing Na+ in the enlarged vacuoles in root hair cells. The upregulation of AtNHX1 transcript levels by NaCl is reduced in abil-1, aba2-1, and aba3-1 but not in abi2-1, sos1, sos2, or sos3 mutants of Arabidopsis. ABAinduced AtNHX1 expression is also decreased in abil-1 but not in abi2-1. This evidence suggests that salt stress up-regulates AtNHX1 expression transcriptionally and that the up-regulation is partially dependent on ABA biosynthesis and ABA signaling through ABI1 (Shi and Zhu, 2002). With a homologous gene region a Na+/H+ antiporter gene has been isolated from a halophytic plant, Atriplex gmelini, and named AgNHX1 (Hamada et al., 2001). The isolated cDNA is 2607 bp in length and contains one open reading frame, which comprises 555 amino acid residues with a predicted molecular mass of 61.9 kDa. The amino acid sequence of the AgNHX1 gene shows more than 75% identity with those of the previously isolated NHX1 genes from the glycophytes A. thaliana and Oryza sativa. The migration pattern of AgNHX1 is shown to correlate with H+-pyrophosphatase and not with P-type H+-ATPase, suggesting the localization of AgNHX1 in a vacuolar membrane. Induction of the AgNHX1 gene has been observed by salt stress at both mRNA and protein levels. The expression of the AgNHX1 gene in the yeast mutant, which lacks the vacuolar-type Na+/H+ antiporter gene (NHX1) and has poor viability under the high-salt conditions, shows partial complementation of the NHX1 functions. It has been suggested that the AgNHX1 products have important roles in salt tolerance (Hamada et al., 2001). Experimental evidence implicates Ca2+ function in salt adaptation. Externally supplied Ca2+ reduces the toxic effects of NaCl, presumably by facilitating higher K+/Na+ selectivity (Liu and Zhu, 1997; Lauchli and Schubert, 1989). High salinity also results in increased cytosolic Ca2+ that is transported from the apoplast


and intracellular compartments (Knight et al., 1997). The resultant transient Ca2+ increase potentiates stress signal transduction and leads to salt adaptation (Mendoza et al., 1994; Knight et al., 1997). A prolonged elevated Ca2+ level may, however, also pose a stress; if so, reestablishment of Ca2+ homeostasis is a requisite. Other mechanisms of salt regulation are salt secretion and selective salt accumulation or exclusion. Salt secretion occurs through development of unique cellular structures called salt glands. These salt glands secrete salt (especially NaCl) from leaves and maintain internal ion concentration at lower level (Hogarth, 1999). Salt exclusion occurs through roots to regulate the salt content of their leaves in many halophytes (Levitt, 1980). Selective accumulation of ions or solutes enables the plants to make osmotic adjustments, which occur through mass action, and results in increased water retention and/or sodium exclusion. 2.3. Induced biosynthesis of compatible solutes To accommodate the ionic balance in the vacuoles, cytoplasm accumulates low-molecular-mass compounds termed compatible solutes because they do not interfere with normal biochemical reactions (Yancey et al., 1982; Ford, 1984; Ashihara et al., 1997; Hasegawa et al., 2000; Zhifang and Loescher, 2003); rather, they replace water in biochemical reaction. With accumulation proportional to the change of external osmolarity within species-specific limits, protection of structures and osmotic balance supporting continued water influx (or reduced efflux) are accepted functions of osmolytes (Hasegawa et al., 2000). These compatible solutes include mainly proline (Khatkar and Kuhad, 2000; Singh et al., 2000), glycine betaine (GB) (Rhodes and Hanson, 1993; Khan et al., 2000a; Wang and Nil, 2000), sugars (Kerepesi and Galiba, 2000; Bohnert and Jensen, 1996; Pilon-Smits et al., 1995), and polyols (Ford, 1984; Popp et al., 1985; Orthen et al., 1994; Bohnert et al., 1995). Polyols make up a considerable percentage of all assimilated CO2 and serve several functions: as compatible solutes, as low-molecular-weight chaperones, and as scavengers of stress-induced oxygen radicals (Smirnoff and Cumbes, 1989; Bohnert et al., 1995). Polyols are classified as acyclic (e.g., manitol) and cyclic (e.g., pinitol). Mannitol, a sugar alcohol that may serve as a compatible solute to cope with salt stress, is synthesized via the action of a mannose-6-phosphate reductase (M6PR) in celery (Zhifang and Loescher, 2003). In contrast to previous approaches that have used a bacterial gene to engineer mannitol biosynthesis in plants and other organisms, A. thaliana, a nonmannitol-producer, has been transformed with the celery leaf M6PR gene under control of the CaMV 35S promotor. In all independent Arabidopsis M6PR

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transformants, mannitol accumulates throughout the plants in amounts ranging from 0.5 to 6 mmol g1 fresh weight. A novel compound, not found in either celery or Arabidopsis, 1-O-beta-D-glucopyranosyl-D-mannitol, also accumulates in vegetative tissues of mature plants in amounts up to 4 mmol g1 fresh weight but not in flowers and seeds. In the absence of NaCl, all transformants are phenotypically the same as the wild type; however, in the presence of NaCl, mature transgenic plants shows a high level of salt tolerance, i.e., growing, completing normal development, flowering, and producing seeds in soil irrigated with 300 mM NaCl in the nutrient solution. These results demonstrate a major role in developing salt-tolerant plants by means of introducing mannitol biosynthesis using M6PR (Zhifang and Loescher, 2003). Pinitol is synthesized from myo-inositol by the sequential catalysis of inositolo-methyltransferase and ononitol epimerase (Bohnert and Jensen, 1996). Polyols function in two ways that are difficult to separate mechanistically: osmotic adjustment and osmoprotection. In osmotic adjustment, they act as osmolytes facilitating the retention of water in the cytoplasm and allowing sodium sequestration to the vacuole or apoplast. These osmolytes protect cellular structures by interacting with membranes, protein complexes, or enzymes. These compounds have hydrogen-bonding characteristics that allow them to protect macromolecules from the adverse effects of increasing ionic strength in the surrounding media (Crowe et al., 1992). By tight association with protein and membrane components, they compensate for water loss during stress (Yancey et al., 1982). Those polyols that are nonreducing sugars may also store excess carbon under environmental stress conditions (Vernon et al., 1993). The cyanobacterium Synechocystis sp. PCC 6803 accumulates the compatible solute glucosylglycerol (GG) and sucrose under salt stress (Hagemann and Murata, 2003). The molecular mechanisms for GG synthesis including regulation of the GG-phosphate synthase (ggpS) gene, which encodes GgpS, has been intensively investigated. Experimental evidences shows that GG is important for salt tolerance and thus for the proper division of cells under salt stress conditions in Synechocystis (Hagemann and Murata, 2003). Accumulation of the polyol myo-inositol in leaf tissues of Actidinia takes place under salt stress and accumulation of myo-inositol is negatively correlated to fructose and glucose (Klages et al., 1999). Severe salt stress (4120 mM NaCl) led to a preferential accumulation of D-pinitol in gametophytes of Acrostichum aureum, whereas the sporophyte accumulates D-1-O-methylmuco-inositol (Sun et al., 1999). Carbohydrates such as sugars (glucose, fructose, sucrose, fructans) and starch accumulate under salt stress (Parida et al., 2002). Their major functions are osmoprotection, osmotic adjustment, carbon storage,


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and radical scavenging. Salt stress increases reducing sugars (glucose, fructose), sucrose, and fructans in a number of plants (Kerepesi and Galiba, 2000; Khatkar and Kuhad, 2000; Singh et al., 2000). In Vicia faba salinity decreases soluble and hydrolyzable sugars (Gadallah, 1999). Sugar content increases in some genotypes of rice but also decreases in some genotypes (Alamgir and Ali, 1999). Under salinity, the starch content in roots of rice plants declines and remains unchanged in shoots. A decrease in starch content and an increase in both reducing and nonreducing sugar have been reported in leaves of Bruguiera parviflora (Parida et al., 2002) (Fig. 2). The contents of reducing and nonreducing sugars and the activity of sucrose phosphate synthase increase under salt stress, whereas starch phosphorylase activity decreases (Dubey and Singh, 1999). In leaves of tomato the contents of soluble sugars and total saccharides are significantly increased, but the starch content is not affected significantly by NaCl treatment (Khavarinejad and Mostofi, 1998). Gao et al. (1998) have reported that in tomato (Lycopersicon esculentum L.) salinity enhances sucrose concentration and activity of sucrose phosphate synthase (EC 2.3.1.14) in leaves but decreases the activity of acid invertase (EC 2.3.1.14). It has also been reported that polyphenol level

C

1

2

3

4

M 66.0

43.0

29.0

increases in leaves of B. parviflora by salt stress (Fig. 4) (Parida et al., 2002). A number of nitrogen-containing compounds (NCC) accumulate in plants exposed to saline stress. The most frequently accumulating NCC include amino acids, amides, imino acids, proteins, quaternary ammonium compounds, and polyamines. The specific NCC that accumulate in saline environments varies with plant species. Osmotic adjustment, protection of cellular macromolecules, storage of nitrogen, maintenance of cellular pH, detoxification of the cells, and scavenging of free radicals are proposed functions of these compounds under stress conditions. NCC accumulation is usually correlated with plant salt tolerance (Mansour, 2000). There are several reports of accumulation of free amino acids and other NCC under salt stress. Glycine betaine content increases by salt stress in a number of plants (Khan et al., 1998, 1999, 2000a; Saneoka et al., 1999; Muthukumarasamy et al., 2000; Wang and Nil, 2000). Khan et al. (2000a) have reported that glycine betaine content increases in shoots and does not differ significantly in roots of Haloxylon recurvum under salt stress. Many plants accumulate proline as a nontoxic and protective osmolyte under saline conditions (Lee and Liu, 1999; Khatkar and Kuhad, 2000; Muthukumarasamy et al., 2000; Singh et al., 2000; Jain et al., 2001). It has been reported that proline levels increase significantly in leaves of nonsecretor mangrove B. parviflora (Fig. 3) (Parida et al., 2002) and in saltsecretor mangrove Aegiceras corniculatum (Parida et al., unpublished data). The most abundant amino acids (cysteine, arginine, methionine), constituting about 55% of total free amino acid content, are reduced in NaCltreated plants of wheat, but valine, isoleucine, aspartic acid, and proline increase in response to NaCl stress and NaCl-treated wheat seedlings show 1.6-fold increase in total free amino acids compared to the control (Elshintinawy and Elshourbagy, 2001). In Zea mays,

20.1 14.3

Fig. 2. Changes in protein profle in leaf of B. parviflora after 7, 14, and 30 days recovery from salt stress. Lanes C and 1 represent proteins extracted from control and 400 mM NaCl-treated (45 day) leaves. Lanes 2, 3, and 4 represent proteins extracted from leaves after 7, 14 and 30 days of desalinization, respectively. Lane M represents the molecular weight marker. The boldface arrow on the left indicates the 23-kDa polypeptide, which reappears after desalinization (from Parida et al., 2004c).

Fig. 3. Effects of NaCl stress on proline level in B. parviflora measured as a function of days of NaCl treatment. Values are mean7SE (from Parida et al., 2002).

Table 1 Responses to salt-stress-accumulating products and their function(s) in conferring tolerance Suggested function(s)

References

Ions

Sodium, chloride

Osmotic adjustment Potassium exclusion/Export

Blumwald et al. (2000); Hasegawa et al. (2000); Nilu et al. (1995) Koyro (2000) Khan et al. (2000a, b)

Proteins

Osmotin SOD/Catalase

Pathogenesis-related proteins Osmoprotection Radical detoxification

Singh et al. (1987); King et al. (1988) Bohnert and Jensen (1996) Bohnert and Jensen (1996); Allen et al (1997); Hernandez et al. (2000)

Amino acids

Proline Ectoine

Osmotic adjustment Osmoprotection

Khatkar and Kuhad (2000); Singh et al. (2000); Lin et al. (2002) Lippert and Galinski (1992)

Sugars

Glucose, fructose, sucrose Fructans

Osmotic adjustment Osmoprotection, carbon storage

Kerepesi and Galiba (2000) Bohnert and Jensen (1996); Pilon-Smits et al. (1995)

Polyols

Acyclic (e.g., manitol) Cyclic (e.g., pinitol)

Carbon storage, osmotic adjustment Osmoprotection, osmotic adjustment Retention of photochemical efficiency of PSII Radical scavenging

Popp et al. (1985); Bohnert et al. (1995) Ford (1984); Bohnert et al. (1995) Sun et al (1999) Smirnoff and Cumbes (1989); Orthen et al. (1994)

Polyamines

Spermine, spermidine

Ion balance, chromatin protection

Tiburico et al. (1993); SantaCruz et al. (1998)

Quaternary amines Glycine betaine b-Alanine betaine, Dimethyl-sulfonio propionate, Choline-o-sulfate Trigonelline Pigments

Osmoprotection Khan et al. (2000a); Wang and Nil (2000) Preservation of thylakoid and plasma membrane integrity Rhodes and Hanson (1993) Osmoprotection Rhodes and Hanson (1993) Osmoprotection Hanson (1998); Trossat et al. (1998) Osmoprotection Nuccio et al. (1999) Rajasekaran et al. (2001)

Carotenoids, anthocyanins, betalaines Protection against photoinhibition

Foyer et al. (1994); Adams et al. (1992a); Kennedy and De Fillippis (1999)

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with increasing external salt concentration, amino acids and quaternary ammonium compounds accumulate in leaves and roots, with concentrations in leaves exceeding those in roots (AbdElBaki et al., 2000). Proline accumulates in leaves, stems, and roots of Pringlea antiscorbutica (kerguelen cabbage) and this osmolyte accumulate at a 2–3 higher concentration in the cytoplasm than in the vacuole (Aubert et al., 1999). Mattioni et al. (1997) have reported that most of the amino acids show an increase with the induction of water or salt stress in durum wheat (Triticum durum), but proline increases more markedly than other amino acids and the activity of the enzyme delta (1)-pyrroline5-carboxylate reductase (EC 1.5.1.2) involved in proline biosynthesis is enhanced during both water and salt stress, while the activity of proline dehydrogenase (EC 1.5.1.2) involved in proline catabolism is inhibited only during salt stress. These results indicate that synthesis de novo is the predominant mechanism in proline accumulation in durum wheat. In mulberry, free amino acids increase at low salinity and decrease at high salinity and glycine betaine accumulates more than proline (Agastian et al., 2000). Proline, quaternary ammonium, and tertiary sulfonium osmolytes are zwitter ions at physiological pH. Although they are ionic, they have no net charge. Their osmotic function is due to their unique chemistry. Experimental observations shows that transformation of plants with the codA gene for choline oxidase enhances tolerance of the transgenic plants to salt stress at the reproductive stage as a result of the accumulation of GB in the reproductive organs (Sulpice et al., 2003). The compatible solute N-epsilon-acetyl-beta-lysine is unique to methanogenic archaea and is produced under salt stress only (Pfluger et al., 2003). However, the molecular basis for the salt-dependent regulation of Nepsilon-acetyl-beta-lysine formation is unknown. Genes potentially encoding lysine-2, 3-aminomutase (abl4) and beta-lysine acetyltransferase (ablB), which are assumed to catalyze N-epsilon-acetyl-beta-lysine formation from alpha-lysine, have been identified on the chromosomes of the methanogenic archaea Methanosarcina mazei Gol, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis (Pfluger et al., 2003). Table 1 provides examples of plant products whose action correlates with metabolic functions that are known or assumed to enable plants to cope with salt tolerance. A common feature of compatible solutes is that these compounds can accumulate to high levels without disturbing intracellular biochemistry (Bohnert and Jensen, 1996). Compatible solutes have the capacity to preserve the activity of enzymes that are in saline solutions. These compounds have minimal effect on pH or charge balance of the cytosol or lumenal compartments of organelles. The synthesis of compatible

osmolytes is often achieved by diversion of basic intermediary metabolites into unique biochemical reactions. Often, stress triggers this metabolic diversion. For example, higher plants synthesize glycine betaine from choline by two reactions that are catalyzed in sequence by choline monooxygenase and betaine aldehyde dehydrogenase (Rhodes and Hanson, 1993). 2.4. Induction of antioxidative enzymes Salt stress is complex and imposes a water deficit because of osmotic effects on a wide variety of metabolic activities (Greenway and Munns, 1980; Cheeseman, 1988). This water deficit leads to the formation of reactive oxygen species (ROS) such as superoxide (Od 2 ), hydrogen peroxide (H2O2), hydroxyl radical (dOH) (Halliwell and Gutteridge, 1985), and singlet oxygen (1O2) (Elstner, 1987). These cytotoxic activated oxygen species can seriously disrupt normal metabolism through oxidative damage to lipids (Fridovich, 1986; Wise and Naylor, 1987) and to protein and nucleic acids (Fridovich, 1986; Imlay and Linn, 1988). Since internal O2 concentrations are high during photosynthesis (Steiger et al., 1977), chloroplasts are especially prone to generate activated oxygen species (Asada and Takahashi, 1987). Once produced, Od will rapidly dismutate, either 2 enzymatically or nonenzymatically, to yield H2O2 and O2. In addition, H2O2 may interact in the presence of certain metal ions or metal chelates to produce highly reactive dOH (Imlay and Linn, 1988). In varying degrees, present day plants possess a number of antioxidants that protect against the potentially cytotoxic species of activated oxygen. The metalloenzyme superoxide dismutase (SOD; EC 1.15.1.1) converts Od 2 to H2O2. Catalase and a variety of peroxidases (Chang et al., 1984) catalyze the breakdown of H2O2. Although catalase is apparently absent in the chloroplast, H2O2 can be detoxified in a reaction catalyzed by an ascorbatespecific peroxidase often present in high levels in this organelle (Chen and Asada, 1989) through the ascorbate–glutathione cycle (Halliwell and Gutteridge, 1986; Asada, 1992). Both ascorbate and glutathione have been reported in millimolar concentrations within the chloroplast (Halliwell, 1982). Ascorbate can also be oxidized by direct reaction with Od 2 or by serving as a reductant of the a-chromoxyl radical of oxidized a-tocopherol (Foyer et al., 1991). The thylakoid membranes are rich in a-tocopherol which disrupts lipid peroxidation reactions not only by reacting with Od but also by scavenging 2 hydroxyl, peroxyl, and alkoxyl radicals (Halliwell, 1987). When plants are subjected to environmental stress conditions such as high light intensity, temperature extremes, drought, high salinity, herbicide treatment, or mineral deficiencies, the balance between the production of reactive oxygen species and the quenching activity of the antioxidants is upset, often resulting in oxidative


damage (Harper and Harvey, 1978; Dhindsa and Matowe, 1981; Wise and Naylor, 1987; Spychalla and Desborough, 1990). Plants with high levels of antioxidants, either constitutive or induced, have been reported to have greater resistance to this oxidative damage (Harper and Harvey, 1978; Dhindsa and Matowe, 1981; Wise and Naylor, 1987; Spychalla and Desborough, 1990). The activities of the antioxidative enzymes such as catalase (CAT), ascorbate peroxidase (APX), guaicol peroxidase (POD), glutathione reductase (GR), and superoxide dismutase increase under salt stress in plants and a correlation of these enzyme levels and salt tolerance exists (Gossett et al., 1994; Hernandez et al., 1995, 2000; Sehmer et al., 1995; Kennedy and De Fillippis, 1999; Sreenivasulu et al., 2000; Benavides et al., 2000; Lee et al., 2001; Mittova et al., 2002, 2003). In soybean root nodules ascorbate peroxidase, catalase, and glutathione reductase activities decrease under salt stress, while superoxide dismutase and reduced glutathione increase and malondialdehyde and total protein remain unchanged (Comba et al., 1998). Transgenic plants have been generated to probe the effects of ROS scavenging on salinity stress tolerance, based on observations of gene expression changes in salt-stressed plants. A putative phospholipid hydroperoxide glutathione peroxidase (PHGPX) transcript increases during salt stress in Arabidopsis and citrus (Gueta-Dahan et al., 1997; Sugimoto and Sakamoto, 1997); also in citrus, transcripts and enzyme activities of Cu/Zn-SOD, glutathione peroxidase, and cytosolic APX increase (Holland et al., 1993; Gueta-Dahan et al., 1997). Catalase-deficient (antisense) tobacco shows enhanced sensitivity to oxidative stress under conditions of light and salinity (Willkens et al., 1997). ROS scavenging as an important component of abiotic stress responses is documented by mutant analysis. The ascorbic-acid-deficient Arabidopsis semidominant soz1 accumulates only 30% of ascorbate compared with wild type, and plants show significantly higher sensitivity to oxidative stress conditions (Conklin et al., 1996). Further support comes from the study of transgenic models, which have been generated to study antioxidant defenses (Orr and Sohal, 1992; Creissen et al., 1996; Allen et al., 1997; Noctor and Foyer, 1998; Smirnoff, 1998). Overexpression of genes leading to increased amounts and activities of mitochondrial Mn-SOD, FeSOD, chloroplastic Cu/Zn-SOD, bacterial catalase, and glutathione-S-transferase (GST)/glutathione peroxidase (GPX) can increase the performance of plants under stress (Bowler et al., 1991; Gupta et al., 1993a, b; Van Camp et al., 1996; Shikanai et al., 1998; Roxas et al., 2000). A cDNA clone encoding a cytosolic SOD has been isolated from a cDNA library constructed from poly(A)+ RNA from epicotyls of 5-day-old Cicer arietinum, L. etiolated seedlings after a differential screening to select clones whose expression decreases

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with epicotyl growth (Hernandez et al., 2002). Analysis of its deduced amino acid sequence shows all the typical structural motifs of plant cytosolic SODs (EC 1.15.1.1.). The expression of this clone is always higher in young and growing tissues than in old and storage tissues and diminishes throughout the development of the seedlings. Cytosolic Cu/Zn-SOD activity is also higher in radicles and younger internodes. Under stress conditions only cold increases the gene expression whereas the activity is clearly increased by a saline medium (Hernandez et al., 2002). To analyze the potential of the active oxygenscavenging system of the cytosol in leaves of saltstressed mangrove B. gymnorrhiza, Takemura et al. (2002) have isolated a full-length cDNA encoding a 153amino-acid sequence of cytosolic Cu/Zn-SOD and a partial cDNA encoding catalase. Northern blot analyses show that the transcript level of cytosolic Cu/Zn-SOD increases after 1 and 5 days of NaCl treatment, but no significant change occurs in the expression of the catalase gene. The transcript of cytosolic Cu/Zn-SOD is also induced by mannitol treatment. This suggests that the increase in cytosolic Cu/Zn-SOD 1 day after NaCl treatment is a response to osmotic stress. After 5 days of treatment with NaCl, the transcript level of cytosolic Cu/Zn-SOD increases in young and mature leaves but not in old leaves. Expression of the cytosolic Cu/Zn-SOD gene is induced by exogenous abscisic acid, while the catalase gene is induced by application of 2-chloroethylphosphonic acid, which is a generator of ethylene. The results from this study suggest that salt stress leads to the generation of superoxide in the cytosol and that the oxygen-scavenging system in the cytosol contributes to the salt tolerance capacity of B. gymnorrhiza (Takemura et al., 2002). 2.5. Induction of plant hormones High salt concentration triggers an increase in levels of plant hormones such as ABA and cytokinins (Thomas et al., 1992; Aldesuquy, 1998; Vaidyanathan et al., 1999). Abscisic acid is responsible for the alteration of salt-stress-induced genes (de Bruxelles et al., 1996). ABA-inducible genes are predicted to play an important role in the mechanism of salt tolerance in rice (Gupta et al., 1998). Salt stress results in increased levels of ABA, aminocyclopropane-1-carboxylic acid, and ethylene production in Citrus sinensis (GomezCadenas et al., 1998). ABA has been found to alleviate the inhibitory effect of NaCl on photosynthesis, growth and translocation of assimilates (Popova et al., 1995). ABA also promotes switch from C3 to crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum under salt stress (Thomas et al., 1992). ABA promotes stomatal closure by rapidly altering ion fluxes in guard cells under stress conditions. Other ABA actions involve



modifications of gene expression, and the analysis of ABA-responsive promoters has revealed diversity of potential cis-acting regulatory elements. The nature of the ABA receptors(s) remains unknown. In contrast, combined biophysical, genetic, and molecular approaches have led to considerable progress in the characterization of more downstream signaling elements. In particular, substantial evidence points to the importance of reversible protein phosphorylation and modification of cytosolic calcium levels and pH as intermediates in ABA signal transduction (Leung and Giraudat, 1998). Experimental evidence shows that the increase of Ca2+ uptake is associated with the rise of ABA under salt stress and thus contributes to membrane integrity maintainance, which enables plants to regulate uptake and transport under high levels of external salinity in the longer term (Chen et al., 2001). It has been reported that ABA reduces ethylene release and leaf abscission under salt stress in citrus probably by decreasing the accumulation of toxic Cl ions in leaves (GomezCadenas et al., 2002). Salt tolerance of facultatively halophytic Lophopyrum elongatum and the closely related but less salt tolerant wheat T. aestivum L. is enhanced when plants are allowed to gradually acclimate to salt stress in comparison to when they are suddenly shocked (Noaman et al., 2002). This acclimation to salt stress is regulated by ABA; a pretreatment with ABA substitutes for the acclimation and increases tolerance of salt shock. The ABA-induced acclimation is rapid and coincides with enhanced expression of the early salt induced (ESI) genes in the roots. L. elongatum tolerates salt shock better than wheat and its genome confers greater tolerance of salt shock in their amphiploid than wheat. The tolerance of salt shock is principally controlled by chromosome 3E in the L. elongatum genome. Wheat homologous chromosomes 3A and 3D also control salt shock response. It is speculated that chromosome 3 in both species mediates salt shock response via ABA (Noaman et al., 2002). Jasmonates also have important roles in salt tolerance. Experimental evidence shows that salt-tolerant tomato cultivars have higher levels of jasmonates than salt-sensitive cultivars (Hilda et al., 2003). Jasmonates are generally considered to mediate signaling, such as defense responses, flowering, and senescence. However, factors involved in the jasmonate signal-transduction pathway remain unclear. To clarify the functions and signaling mechanisms of jasmonates on a genomewide level, Sasaki et al. (2000) have adopted a cDNA macroarray technique. By analyzing the data from the cDNA macroarray, many function-known and -unknown genes have been identified as MeJA-responsive genes, and the profiles of expression show good agreement with Northern blot analysis (Sasaki et al., 2000).

2.6. Change in photosynthetic pathway Salt stress inhibits photosynthesis by reducing water potential. So the main aim of salt tolerance is to increase water use efficiency under salinity. To this end, facultative halophytic plants such as M. crystallinum shift their C3 mode of photosynthesis to CAM (Cushman et al., 1989). This change allows the plant to reduce water loss by opening stomata at night, thus decreasing transpiratory water loss under prolonged salinity conditions. There is also a shift from the C3 to the C4 pathway in response to salinity in salt-tolerant plant species such as Atriplex lentiformis (Zhu and Meinzer, 1999). 2.7. Molecular mechanism of salt tolerance 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). Selective examples of genes/proteins induced by salt stress are given in Table 2. These genes can be classified in functional groups (Table 3) related to their physiologic or metabolic function predicted from sequence homology with known proteins. Salt tolerance is a multigenic trait and a number of genes categorized into different functional groups are responsible for encoding salt-stress proteins: (i) genes for photosynthetic enzymes, (ii) genes for synthesis of compatible solutes, (iii) genes for vacuolar-sequestering enzymes, and (iv) genes for radical-scavenging enzymes. Most of the genes in the functional groups have been identified as salt inducible under stress conditions. Other genes have been detected by a salt-hypersensitivity assay in Arabidopsis, which led to the identification of mutants in potassium uptake as being critical in salt sensitivity (Wu et al., 1996). However, other physiological systems may be equally limiting under stress conditions and mutants in these physiological pathways could lead to increased salt toxicity and would affect survival in a negative manner. Transcript regulation in response to high salinity has been investigated for salt-tolerant rice (var Pokkali) with microarrays including 1728 cDNAs from libraries of salt-stressed roots (Kawasaki et al., 2001). NaCl at 150 mM reduces photosynthesis to one-tenth of the prestress value within minutes. Hybridizations of RNA to microarray slides were probed for changes in transcripts from 15 min to 1 week after salt shock. Beginning 15 min after the shock, Pokkali shows up-regulation of transcripts. Approximately 10% of the transcripts in Pokkali are significantly up- or down-regulated within 1 h of salt stress. The initial differences between control and stressed plants continue for hours but become less pronounced as the plants adapted over time. The

Table 2 Selective examples of genes/proteins induced by salt stress Characteristic feature(s)

Reference

Arabidopsis thaliana

Sal 1

Quintero et al. (1996)

Brassica napus

Bnd 22

Citrus cinensis Dunaliella salina Hordeum ulgare

Salt associated 23 to 25 kDa protein P 150 26 and 27 kDa proteins (salt-induced polypeptides SIP S1–S4) hva 1 TAS-12 le-16 gene ppc-1 and ppc-2

Induced by salt stress, overexpression in Arabidopsis or yeast overcomes Na+ and Li+ toxicity, homologous to hal 1 of yeast 22 kDa protein, level increased by progessive or rapid water stress and salinity Induced by salt stress, ABA, and water stress 150 kDa protein, induced by salt stress, de novo synthesized protein Salt stress induced S1–S4 polypeptides but water deficit did not induce S2 polypeptides Induced by ABA, drought, NaCl, cold, and heat treatment Salt and water stress induced lipid transfer protein Induced by drought, PEG, salinity, cold, and heat stress Encodes PEP carboxylase, induced by salt and water stress, exogenous ABA is a poor substitute for NaCl to induce it

Lycopersicom esculantum Mesembryanthemum crystallinum

Isogenes lmt 1 Inps 1 Nicotiana tabacum

26- and 43-kDa polypeptides 58-, 37-, 35.5-, 34-, 26-, 21-, 19.5-, and 18-kDa polypeptides 30-kDa polypeptide Vitronectin and fibronectin-like proteins Osmotin

O. sativa

RAB21

salT em

Reviron et al. (1992) Benhayyim et al. (1993) Sadaka et al. (1991) Hurkman and Tanaka (1988) Hong et al. (1992) Torres-Schumann et al. (1992) Plant et al. (1991) Cushman et al. (1989)

Encodes myo-inositol o-methyl transferase 1; induced by NaCl and osmotic stress Encodes myo-inositol 1-phosphate synthase; shows significant homology to corresponding genes in plants and yeast Levels increase in both NaCl, and PEG-induced water stress adapted cells but are not detected in unadapted Increased levels with increased NaCl tolerance

Vernon and Bohnert (1992)

Heat shock at 38 1C induces cross tolerance to salt stress Found in membranes and cell wall of NaCl-adapted cells

Harrington and Alm (1988) Zhu et al. (1993)

26-kDa protein, protein level enhanced in both NaCl- and PEG-induced water stress adapted cells but not in unadapted cells Induced when plants are subjected to water stress, rab21 mRNA and protein accumulate in rice embryos, leaves, roots, and callus derived suspension cells upon treatment with NaCl and/or ABA mRNA accumulates rapidly in shoots and roots of mature seedlings with ABA salts, PEG, NaCl, and KCl; no induction in leaf lamina Induced by ABA and salt stress, salt interacts synergistically with ABA

Singh et al. (1987)

Ishitani et al. (1996) Singh et al. (1985) Singh et al. (1985)

Mundy et al. (1990)

Claes et al. (1990) Bostock and Quatrano (1992)

The names of various genes and proteins have been by and large reproduced here as per the original publications of the authors.

ARTICLE IN PRESS

Salt responsive genes/proteins


Plant species

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Table 3 Functional groups of genes/proteins activated in salt stress with potential for providing tolerance (reproduced from Winicov, 1998)

Table 4 Differences between halophytes and glycophytes with respect to salt tolerance mechanism

1 2 3 4 5 6 7 8 9

Halophytes

Glycophytes

1.

1. Higher.

Carbon metabolism and energy production/photosynthesis Cell wall/membrane structural components Water channel proteins Ion transport Oxidative stress defenses Detoxifying enzymes Proteinases Proteins involved in signaling Transcription factors

2. 3. 4. 5. 6.

interpretation of an adaptive process is supported by the similar analysis of salinity-sensitive rice (var IR29), in which the immediate response exhibited by Pokkali is delayed and later results in down-regulation of transcription and death. The up-regulated functions observed with Pokkali at different time points during stress adaptation change over time. Increased protein synthesis and protein turnover are observed at early time points, followed by the induction of known stressresponsive transcripts within hours and the induction of transcripts for defense-related functions later. After 1 week, the nature of up-regulated transcripts (e.g., aquaporins) indicates recovery (Kawasaki et al., 2001). Acclimation of microorganisms to environmental stress is closely related to the expression of various genes (Kanesaki et al., 2002). It has been reported that salt stress and hyperosmotic stress have different effects on the cytoplasmic volume and gene expression in Synechocystis sp. PCC 6803 (Kanesaki et al., 2002). DNA microarray analysis indicates that salt stress strongly induces the genes for some ribosomal proteins. Hyperosmotic stress strongly induces the genes for 3ketoacyl-acyl carrier protein reductase and rare lipoprotein A. Genes whose expression is induced both by salt stress and by hyperosmotic stress include those for heat shock proteins and the enzymes for the synthesis of glucosylglycerol. It has also been reported by Kanesaki et al. (2002) that each kind of stress induces a number of genes for proteins of unknown function. Findings of Kanesaki et al. (2002) suggests that Synechocystis sp. recognizes salt stress and hyperosmotic stress as different stimuli, although mechanisms common to the responses to each form of stress might also contribute to gene expression. 2.8. Salinity effect: halophytes vs. glycophytes The salt tolerance mechanism is a multigenic trait and therefore biochemical pathways leading to products or processes that improve salt tolerance are likely to act additively and probably synergistically. So the advantage of halophytes over glycophytes can be through more efficient performance of a new basic biochemical

Salinity has a smaller effect upon the extent to which stomata limit photosynthesis. High water use efficiency. Low internal CO2 concentration. Low light saturated photosynthetic rate. Efficient accumulating solutes. Low level of Na+ and Cl ions in the cytoplasm and chloroplast.

2. Comparatively less. 3. Comparatively less. 4. It is less. 5. Less efficient. 6. It is higher.

tolerance mechanism. Table 4 summarizes differences in halophytes and glycophytes with respect to salt tolerance mechanism.

3. Salinity effects on plants Salinity of soil and water is caused by the presence of excessive amounts of salts. Most commonly, high Na+ and Cl cause the salt stress. Salt stress has threefold effects; viz. it reduces water potential and causes ion imbalance or disturbances in ion homeostasis and toxicity. This altered water status leads to initial growth reduction and limitation of plant productivity. Since salt stress involves both osmotic and ionic stress (Hagemann and Erdmann, 1997; Hayashi and Murata, 1998), growth suppression is directly related to total concentration of soluble salts or osmotic potential of soil water (Flowers et al., 1977; Greenway and Munns, 1980). The detrimental effect is observed at the whole-plant level as death of plants or decrease in productivity. Suppression of growth occurs in all plants, but their tolerance levels and rates of growth reduction at lethal concentrations of salt vary widely among different plant species. Although the change in water status is the cause of growth suppression, the contribution of subsequent processes to inhibition of cell division and expansion and acceleration of cell death has not been well documented (Hasegawa et al., 2000). Salt stress affects all the major processes such as growth, photosynthesis, protein synthesis, and energy and lipid metabolism. These are discussed under separate headings. 3.1. Effects of salinity on growth Salinity stress results in a clear stunting of plants (Hernandez et al., 1995; Cherian et al., 1999; Takemura et al., 2000). The immediate response of salt stress is reduction in the rate of leaf surface expansion leading to


cessation of expansion as salt concentration increases (Wang and Nil, 2000). Salt stress also results in a considerable decrease in the fresh and dry weights of leaves, stems, and roots (Hernandez et al., 1995; AliDinar et al., 1999; Chartzoulakis and Klapaki, 2000). The optimum growth of plants is obtained at 50% seawater and declines with further increases in salinity in Rhizophora mucronata (Aziz and Khan, 2001). Fresh and dry weights of plants increase with an increase in salinity in Salicornia rubra while the optimal growth occurrs at 200 mM NaCl and the growth declines with a further increase in salinity (Khan, 2001). In Raphanus sativus (radish) total plant dry weight decreases at higher salinities and about 80% of the growth reduction at high salinity can be attributed to reduction of leaf area expansion and hence to reduction of light interception. The remaining 20% of the salinity effect on growth is most likely explained by a decrease in stomatal conductance. The small leaf area at high salinity is related to a reduced specific leaf area and increased tuber/shoot weight ratio and the latter can be attributed to tuber formation starting at a smaller plant size at high salinity (Marcelis and VanHooijdonk, 1999). Kurban et al. (1999) have reported that in Alhagi pseudoalhagi (a leguminous plant), total plant weight increases at low salinity (50 mM NaCl) but decreases at high salinity (100 and 200 mM NaCl). Khan et al. (1999) have reported that when Halopyrum mucronatum (a perennial grass found on the coastal dunes of Karachi, Pakistan) is treated with 0, 90, 180, and 360 mM NaCl in sand culture, fresh and dry mass of roots and shoots peaks at 90 mM NaCl, a further increase in salinity inhibits plant growth, ultimately resulting in plant death at 360 mM NaCl, and maximum succulence is noted at 90 mM NaCl. Experimental evidence shows that in a salt nonsecretor mangrove B. parviflora the plant growth is optimal at 100 mM NaCl under hydroponic culture, whereas further increase in NaCl concentration retards plant growth (Table 5) and 500 mM NaCl is found to be lethal in this species (Parida et al., 2004a). On the other hand a salt secretor mangrove Aegiceras corniculatum can tolerate upto 250 mM NaCl and 300 mM is found lethal in this case (Mishra and Das, 2003). Increasing salinity is accompanied by significant reductions in

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shoot weight, plant height, number of leaves per plant, root length, and root surface area per plant in tomato (Mohammad et al., 1998). Increased NaCl levels results in a significant decrease in root, shoot, and leaf growth biomass and increase in root/shoot ratio in cotton (Meloni et al., 2001). 3.2. Effects of salinity on water relations Water potential and osmotic potential of plants become more negative with an increase in salinity, whereas turgor pressure increases with increasing salinity (Morales et al., 1998; Hernandez et al., 1999; Khan et al., 1999; Meloni et al., 2001; Khan, 2001; Romeroaranda et al., 2001). Leaf water and osmotic potentials and xylem tension increase with an increase in media salinity in R. mucronata (Aziz and Khan, 2001). Leaf osmotic potentials decrease with increases in NaCl in Chrysanthemum and sea aster (Matsumura et al., 1998). Relative water content, leaf water potential, water uptake, transpiration rate, water retention, and water use efficiency decrease under short-term NaCl stress in jute (Chaudhuri and Choudhuri, 1997). Water potential, osmotic potential, and stomatal conductance become more negative with an increase in salinity, while pressure potential decreases with increasing salinity in the halophytic perennial grass Urochondra setulosa (Trip.) (Gulzar et al., 2003). With increasing salt concentration, leaf water potential and evaporation rate decrease significantly in the halophyte S. salsa while there are no changes in leaf relative water content (Lu et al., 2002). Leaf water potential and osmotic potential decline depending on the osmotic potential of the rooting medium and the mode of stress imposition. A greater decline in osmotic potential compared with the total water potential led to turgor maintenance in plants under progressive or prolonged NaCl stress (Rajasekaran et al., 2001). 3.3. Effects of salinity on leaf anatomy Salinity causes increases in epidermal thickness, mesophyll thickness, palisade cell length, palisade diameter, and spongy cell diameter in leaves of bean,

Table 5 Growth of B. parviflora after 45 days in nutrient solution containing 0–400 mM NaCl [NaCl] (mM)

Plant height (cm/plant)

Leaf area (cm2/ plant)

Fresh matter of plant (g/plant)

Dry matter of plant (g/plant)

Water content per unit leaf area (gm2)

0 100 200 400

24.070.62a 25.570.82a 23.270.55a 22.570.91a

54.970.35a 58.970.90b 46.270.62c 44.670.55c

8.3870.51a 8.7270.35a 8.1170.25a 7.0270.20a

1.9470.05a 2.0470.04a 1.7770.04a 1.3470.05a

301.175.51a 302.072.70a 319.372.35b 324.471.85c

Values are mean7SE of three independent experiments. Different letters next to values indicate statistically different means at Pp0:05 (Source: Parida et al., 2004a).



cotton, and Atriplex (Longstreth and Nobel, 1979). In contrast both epidermal and mesophyll thickness and intercellular spaces decrease significantly in NaCltreated leaves of the mangrove B. parviflora (Parida et al., 2004a). Salinity also reduces intercellular spaces in leaves (Delphine et al., 1998). Salt stress causes (1) vacuolation development and partial swelling of endoplasmic reticulum, (2) decrease in mitochondrial cristase and swelling of mitochondria, (3) vesiculation and fragmentation of tonoplast, and (4) degradation of cytoplasm by the mixture of cytoplasmic and vacuolar matrices in leaves of sweet potato (Mitsuya et al., 2000) (Fig. 4). In leaves of potato salt stress causes rounding of cells, smaller intercellular spaces, and a reduction in chloroplast number (Bruns and Hecht–Buchholz, 1990). In tomato plants salinity causes reduction of plant leaf area and stomatal density (Romeroaranda et al., 2001).

3.4. Effects of salinity on photosynthetic pigments and proteins The chlorophyll and total carotenoid contents of leaves decrease in general under salt stress. The oldest leaves start to develop chlorosis and fall with prolonged period of salt stress (Hernandez et al., 1995, 1999; Gadallah, 1999; Agastian et al., 2000). However, Wang and Nil (2000) have reported that chlorophyll content increases under conditions of salinity in Amaranthus. In Grevilea, protochlorophyll, chlorophylls, and carote-

Fig. 4. Effects of NaCl stress on polyphenol level in B. parviflora measured as a function of days of NaCl treatment. Values are mean7SE (from Parida et al., 2002).

noids are significantly reduced under NaCl stress, but the rate of decline of protochlorophyll and chlorophyll is greater than that of Chl-a and carotenoids. The anthocyanin pigments on the other hand significantly increase in this case with salt stress (Kennedy and De Fillippis, 1999). In leaves of tomato, the contents of total chlorophyll (Chl-a+b), Chl-a, and b carotene decrease by NaCl stress (Khavarinejad and Mostofi, 1998). Under salinity stress, leaf pigments studied in nine genotypes of rice reduce in general, but relatively high pigment levels are found in six genotypes (Alamgir and Ali, 1999). In the cyanobacterium Spirulina platensis a decrease in the phycocyanin/chlorophyll ratio and no significant change in the carotenoid/chlorophyll ratio are observed under salt stress (Lu and Vonshak, 1999). Salinity causes significant decreases in Chl-a, Chl-b, and carotenoid in leaves of B. parviflora (Table 6; Parida et al., 2002). Soluble protein contents of leaves decrease in response to salinity (Alamgir and Ali, 1999; Gadallah, 1999; Wang and Nil, 2000; Muthukumarasamy et al., 2000; Parida et al., 2002). Agastian et al. (2000) have reported that soluble protein increases at low salinity and decreases at high salinity in mulberry. In Rhizobium certain outer membrane proteins of molecular weight 22, 38, 40, 42, 62, and 68 kDa markedly decrease in the presence of salt (Unni and Rao, 2001). SDS–PAGE analysis of proteins in peanut (Arachis hypogaea L.) reveals that plants grown under NaCl show induction (127 and 52 kDa) or repression (260 and 38 kDa) in the synthesis of a few polypeptides (Hassanein, 1999). Salinity induces six new proteins in roots of barley, which are of low molecular weight, 24 to 27 kDa, with an isoelectric point of 6.1 to 7.6. In contrast to roots, five new shoot proteins are induced whose molecular weights and isoelectric points fall within the range of 20–24 kDa and 6.3–7.2, respectively. In addition, salinity also inhibits the synthesis of a majority of shoot proteins (Ramagopal, 1987). In radish (R. sativus L.) salt stress causes accumulation of a 22-kDa protein (pI of 7.5) and its mRNA in the leaves (Lopez et al., 1994). A significant increase in the amount of a protein, whose migration in two-dimensional (2D) gel electrophoresis corresponds to an apparent molecular weight of 23–25 kDa and a pI of 6.1, is observed under NaCl stress in cultured cells derived from Shamuti orange

Table 6 Effects of NaCl stress on photosynthetic pigments in leaves of B. parviflora [NaCl] (mM)

Chl a (mg cm2)

Chl b (mg cm2)

Total Chl

Chl a/b

Total carotenoids (mg cm2)

0 100 200 400

65.4871.2 67.9971.6 40.0671.0 35.5371.3

17.9671.1 20.6571.8 12.0671.3 11.0371.4

83.4471.8 88.6471.5 52.1271.4 46.5671.1

3.64 3.29 3.32 3.20

23.2071.5 23.6571.4 16.5571.1 13.6871.0

4th pair of leaves from shoot top were sampled after 45 days NaCl treatment. The values are mean7SE (n ¼ 6) (Source: Parida et al., 2004a).


(Citrus sinensis L. Osbeck) ovular callus cells (Benhayyim et al., 1993). Yen et al. (1997) have reported the accumulation of five polypeptides with estimated molecular masses of 40, 34, 32, 29, and 14 kDa by SDS and 2D-PAGE in calli of M. crystallinum under NaCl stress and these polypeptides are classified into two distinct groups according to their course of induction: early responsive (40-, 34-, and 29-kDa) and late responsive (32- and 14-kDa) proteins. The disappearance of a 60-kDa polypeptide in response to salinity has been observed in Prosopsis (Munoz et al., 1997). In wheat, the content of a 26-kDa protein increases in NaCl-treated plants, stimulation is more pronounced in roots than in shoots, and, in contrast, the contents of 13and 20-kDa proteins decrease and the 24-kDa protein disappears with NaCl treatment (Elshintinawy and Elshourbagy, 2001). NaCl induces accumulation of four polypeptides with molecular masses of 61, 51, 39, and 29 kDa in maize roots (Tamas et al., 2001). Fractionation of cells followed by SDS–PAGE and 2D-PAGE reveals an increase in the levels of membrane proteins of 39 and 50 kDa and a decrease in the level of a membrane protein of 52 kDa with increasing levels of external NaCl in Rhodobacter sphaeroides. The proteins have been isolated and sequenced, the polypeptide of 50 kDa in the inner membrane is assigned to an ATP synthase beta chain and that of 52 kDa in the outer membrane to a flagellar filament protein, and, as the N terminus of the 39 kDa protein in the outer membrane is blocked, partial proteolysis was carried out and four peptides were sequenced. Each sequence exhibits no significant homology with those available in databases, suggesting that the polypeptide of 39 kDa (named SspA) is a novel salt-stress-induced protein (Xu et al., 2001). It has been reported that salinity causes a decrease in intensity of several protein bands of molecular weight 17, 23, 32, 33, and 34 kDa in B. parviflora (Parida et al., 2004b) and the degree of decrease of these protein bands seems to be roughly proportional to the external NaCl concentration. The most obvious change concerns a 23-kDa polypeptide, which disappears after 45 days of treatment in 400 mM NaCl (Fig. 2). Moreover, the 23-kDa polypeptide, which disappears in B. parviflora under salinity stress, reappears when these salinized seedlings are desalinized (Fig. 2) and these observations suggest the possible involvement of these polypeptides for osmotic adjustment under salt stress in this species (Parida et al., 2004c). 3.5. Effects of salinity on lipids Lipids are the most effective sources of storage energy, they function as insulators of delicate internal organs and hormones, and they play important roles as the structural constituents of most of the cellular membranes (Singh et al., 2002). They also have vital

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roles in the tolerance to several physiological stressors in a variety of organisms including cyanobacteria. The mechanism of desiccation tolerance relies on phospholipid bilayers, which are stabilized during water stress by sugars, especially by trehalose. Unsaturation of fatty acids also counteracts water or salt stress. Hydrogen atoms adjacent to olefinic bonds are susceptible to oxidative attack. Lipids are rich in these bonds and are a primary target for oxidative reactions. Lipid oxidation is problematic as enzymes do not control many oxidative chemical reactions and some of the products of the attack are highly reactive species that modify proteins and DNA (Singh et al., 2002). The lipid content in peanut (A. hypogaea L.) increases at low concentration of NaCl (up to 45 mM) and decreases at higher concentrations (Hassanein, 1999). Wu et al. (1998) have analyzed the changes in lipid composition by NaCl stress in root plasma membrane of salt marsh grass (Spartina patens) and reported that molar percentages of sterols (including free sterols) and phospholipid decreases with increasing salinity, but the sterol/phospholipid ratio is unaffected by NaCl. However, glycolipid shows a statistically significant increase in the total lipid as salinity in the medium is increased and the content of plasma membrane phosphatidylcholine (PC) and phosphatidylethalomine (PE) decrease by salinity, but the PC/PE ratios are not affected by salinity. Plasma membrane vesicles isolated from calli of tomato tolerant to 100 mM NaCl exhibit higher phospholipid and sterol content and lower phospholipid/free sterol ratio and lower double bond index of phospholipid fatty acids (Kerkeb et al., 2001). 3.6. Effects of salinity on ion levels High salt (NaCl) uptake competes with the uptake of other nutrient ions, especially K+, leading to K+ deficiency. Increased treatment of NaCl induces increase in Na+ and Cl and decrease in Ca2+, K+, and Mg2+ levels in a number of plants (Khan et al., 1999, 2000a; Khan, 2001). Salinity enhances the content of Na+, Ca2+, and Cl in Vicia faba and the ratio of K+/Na+ decreases (Gadallah, 1999). In A. pseudoalhagi (a leguminous plant), the leaf Na+ concentration in 200 mM NaCl-treated plants increases to 45 times that of the control and the plants do not die but continue to grow at such a high leaf Na+ concentration (Kurban et al., 1999). In Ulva fasciata, a marine green macro alga, the contents of Na+, K+, and Cl accumulate linearly with increasing salinity. An increase in Na+ and Cl content induces proline accumulation but decreases both the activity of proline dehydrogenase (PDH EC 1.4.3.1; a catabolic enzyme of proline) and the total and water-soluble Ca2+ contents. These results suggest that in U. fasciata a loss of cellular Ca2+ is associated with NaCl induction of proline accumulation via


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Table 7 Effects of salt stress on ion levels in leaves, stems, and root of B. parviflora treated with different concentrations of NaCl for a period of 45 days Plant parts

[NaCl] (mM)

Na

K

Ca

Mg

Fe

Cu

Mn

Cl

Leaf

0 100 200 400

14.4770.7 33.5470.3 38.6370.4 42.1370.2

11.0270.1 12.2670.7 10.8370.6 11.8170.2

12.3170.3 11.4170.2 9.9970.5 7.8170.6

155.9471.9 162.4971.6 145.4671.2 117.1471.4

325.2171.4 309.0771.8 254.1171.5 295.7571.3

31.5070.3 28.5070.2 20.4170.5 18.3770.64

103.1071.6 97.4170.8 53.0670.6 7.1070.6

13.1170.1 38.5370.6 40.2170.4 43.4970.4

Stem

0 100 200 400

9.4870.5 15.8170.7 16.7070.1 21.6770.2

6.0070.5 7.8670.7 3.8170.3 2.7170.2

2.4970.9 2.9170.6 2.2670.7 3.6370.5

65.4170.7 61.5470.3 88.3470.5 298.4170.3

137.171.8 187.070.8 190.570.5 220.471.5

4.3570.7 5.6970.3 2.3970.5 1.4870.9

17.0570.9 21.0770.4 14.0170.7 12.6370.5

7.2870.1 21.9170.3 26.7070.6 38.5470.8

Root

0 100 200 400

7.2170.5 14.4770.3 17.2570.5 26.9670.8

25.4570.4 17.7870.1 15.0370.9 11.2070.5

2.7470.6 2.8970.3 2.4570.8 2.3870.3

205.6870.8 159.5570.4 142.8770.5 116.7870.6

632.171.8 970.971.1 383.970.9 277.870.6

65.9070.9 51.1070.5 28.1071.2 14.5270.7

28.4070.5 37.0070.5 33.0770.3 11.9670.7

6.8570.7 18.3770.3 24.9570.5 31.4770.3

Na, K, Ca, Mg, and Cl content are expressed as mg g d wt1, whereas Fe, Cu, and Mn content are expressed as mg g dwt1. Values are mean7S.E (n ¼ 6) (Source: Parida et al., 2004a).

an inhibition of PDH activity (Lee and Liu, 1999). Salinity stress causes an increase in levels of Na+ and Cl in guava and the highest ion accumulation is found in the leaves followed by the roots; the Ca2+ levels are stable in the roots but decrease in stems and leaves and the K+ content is reduced with increased levels of salinity, particularly in the leaves. On the other hand, Mg2+ levels are not affected by salinity in stems and roots but decrease in the leaves of guava. There is a positive relationship between Na+ and Cl and a negative relationship between Na+ and K+ concentration in roots and leaves. Mg2+ concentration in leaves and roots does not vary with the concentration of Na+, and the concentration of Ca2+ does not vary with that of Na2+ in the leaves but shows an inverse relationship in the roots (Ferreira et al., 2001). We have reported a significant increase in Na+ and Cl content in leaves, stem, and root of the mangrove B. parviflora (Table 7) without any significant alteration of the endogenous level of K+ and Fe2+ in leaves (Parida et al., 2004a). Decreases of Ca2+ and Mg2+ content of leaves have also been reported upon salt accumulation in this species, suggesting increasing membrane stability and decreased chlorophyll content, respectively (Parida et al., 2004a). 3.7. Effects of salinity on antioxidative enzymes and antioxidants Salt stress causes water deficit as a result of osmotic effects on a wide variety of metabolic activities of plants and this water deficit results in oxidative stress because of the formation of reactive oxygen species such as superoxides and hydroxy and peroxy radicals. The reactive oxygen species that are by-products of hyperosmotic and ionic stresses cause membrane disfunction and cell death (Bohnert and Jensen, 1996). The plants

defend against these reactive oxygen species by induction of activities of certain antioxidative enzymes such as catalase, peroxidase, glutathione reductase, and superoxide dismutase, which scavenge reactive oxygen species. There are several reports of increasing activity of antioxidative enzymes. Activities of antioxidative enzymes such as ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and Mn-SOD increase under salt stress in wheat, while Cu/ Zn-SOD remains constant and total ascorbate and glutathione content decrease (Hernandez et al., 2000). In wheat, activities of APX, MDHAR, DHAR, and GR increase in the shoots and decrease in the roots (Meneguzzo and Navarilzzo, 1999). Gossett et al. (1994) have reported that in cotton (Gossypium hirsutum L.) NaCl stress increases the activities of SOD, guaicol peroxidase, and glutathione reductase and decreases the activities of catalase and ascorbate peroxidase. Salt stress also causes decrease in total ascorbate, total glutathione, and a-tocopherol levels in this case. In leaves of rice plant, salt stress preferentially enhances the content of H2O2 and the activities of SOD, APX, and GPX, whereas it decreases catalase activity (Lee et al., 2001). On the other hand, salt stress has little effect on the activity levels of glutathione reductase (Lee et al., 2001). Lechno et al. (1997) have reported that NaCl treatment increases the activities of the antioxidative enzymes catalase and glutathione reductase and the content of the antioxidants ascorbic acid and reduced glutathione but does not affect the activity of SOD in cucumber plants. In radish NaCl stress decreases proline oxidase activity and increases protease, gamma-glutamyl kinase, and ATPase activities (Muthukumarasamy et al., 2000). SOD activity in plant leaves of barley and H+ ATPase activity in plant roots increase by salinity, whereas malondialdehyde (MDA) concentration in


CAT (Umg-1 protein)

30 25

a a ab c

20 15 0mM 100mM 200mM 400mM

10 5 0 0

APX (Umg-1 protein)

(A) 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

GR(U mg-1 protein)

7

14

21

30

0mM 100mM 200mM 400mM

45

b b

a a

0

(B) 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0

7

14

21

30

45 c b a a

0mM 100mM 200mM 400mM

0

7

14

21

30

45

Duration of exposure (days)

(C) 20 SOD(Umg-1 protein)

plant leaves decreases (Liang, 1999). The higher lipoxygenase and antioxidant enzyme activities such as SOD, catalase, ascorbate peroxidase, glutathione reductase, and GST are observed in tomato under NaCl stress (RodriguezRosales et al., 1999). In Grevia ilicifolia, the levels of MDA significantly decline as a result of salt exposure, and this is accompanied by significant increases in both acid protease and neutral protease activities. Four other key enzymes involved in oxdative stress as tissue degradation (catalase, polyphenol oxidase, SOD, and lypoxygenase) are also significantly increased as a result of NaCl treatment (Kennedy and De Fillippis, 1999). Hernandez et al. (1999) have reported that in pea (Pisum sativum cv. Puget) higher concentrations of NaCl (110–130 mM) enhance the activities of cytosolic CuZn-SOD II, chloroplastic CuZn-SOD II, and mitochondrial and/or peroxisomal Mn-SOD. These inductions are matched by increases in the activity of APX and MDHAR. The activities of GR and DHAR are induced only under severe NaCl stress (130–160 mM) and are accompanied by losses in the ascorbate and glutathione pools and ASC/DHA and GSH/GSSG ratios and by increases in GSSG (Hernandez et al., 1999). Salinity induces enhancement of activities of the chloroplast form of Lmyo-inositol 1-phosphate synthase (EC 5.5.4.1) in rice (Raychaudhuri and Majumder, 1996). Experimental evidence shows that salinity causes increases in APX, GPX, GR, and SOD activity and decreases in catalase activity in leaves of B. parviflora (Fig. 5; Parida et al., 2004c). A preferential enhancement of Mn-SOD has also been reported in this species (Parida et al., 2004c). The response of the chloroplastic antioxidant system of the cultivated tomato L. esculentum (Lem) and the wild salt-tolerant related species L. pennellii (Lpa) to NaCl stress has been reported by Mittova et al. (2002). An increase in H2O2 level and membrane lipid peroxidation is observed in chloroplasts of salt-stressed Lem. In contrast, a decrease in these indicators of oxidative stress is characterized by chloroplasts of salt-stressed Lpa plants. This differential response of Lem and Lpa to salinity correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase, ascorbate peroxidase, MDHAR, GST, PHGPX, and several isoforms of nonspecific peroxidases are found in chloroplasts of salt-treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase decreases while in those of salt-stressed Lem it increases. Although total SOD activity slightly increases in chloroplasts of salttreated Lem plants, differentiation between SOD types reveals that only stromal Cu/Zn-SOD activity increases. In contrast, in chloroplasts of salt-treated Lpa plants Fe-SOD activity increases while Cu/Zn-SOD activity remains unchange. These data indicate that the saltdependent oxidative stress and damage suffered by Lem

339

c c

0mM 100mM 200mM 400mM

15

b

10

a 5 0 0

(D)

7 14 21 30 Duration of exposure (days)

45

Fig. 5. Effects of different concentrations of NaCl on activity of catalase (A), ascorbate peroxidase (B), glutathione reductase (C), and superoxide dismutase (D) in leaves of B. parviflora measured as function of days of NaCl treatment. Enzyme activity is expressed as unit per mg protein. Values are mean7SE. Different letters next to the symbols indicate statistically different means as Pp0:01 (from Parida et al., 2004b).

chloroplasts is effectively alleviated in Lpa chloroplasts by the selective up-regulation of a set of antioxidative enzymes (Mittova et al., 2002). The effects of salinity (100 mM NaCl) and different nitrogen sources [NaNO3/ (NH4)2SO4] on the activities and spatial distributions of antioxidative enzymes (glutathione reductase, superoxide dismutase, guaicol peroxidase, and catalase) and GST have been investigated in maize and sunflower seedlings (RiosGonzalez et al., 2002). The antioxidant



enzymes exhibit higher activities in salinity-treated plants. Changes in antioxidant enzyme activities caused by different N sources differ in the two species. Ammonium-fed plants show higher CAT activity in both plant species and higher GR activity in maize and sunflower leaves, with the highest GST activity in maize. POD and SOD activities are lower in both maize and sunflower seedlings and lower GR activity has been observed in maize roots. SOD and POD activities are higher in the mature sections of the root than in the tips. GR activity is higher in the younger parts of the nitratefed plant roots. The antioxidant enzyme activities are higher in the cortex than in the stele of the nodal roots (RiosGonzalez et al., 2002). The response of the antioxidative systems of leaf cell mitochondria and peroxisomes of the cultivated tomato L. esculentum and the wild salt-tolerant related species L. pennellii to NaCl stress has also been investigated (Mittova et al., 2003). Salt-dependent oxidative stress is evident in Lem mitochondria as indicated by their raised levels of lipid peroxidation and H2O2 content whereas their reduced ascorbate and reduced glutathione contents decrease. Concomitantly, SOD activity decreases whereas APX and GPX activities remain at control level. In contrast, the mitochondria of salt-treated Lpa do not exhibit saltinduced oxidative stress. In their case salinity induces an increase in the activities of superoxide dismutase, ascorbate peroxidase, MDHAR, dehydroascorbate reductase, and glutathione-dependent peroxidase. Lpa peroxisomes exhibit increased SOD, APX, MDHAR, and catalase activity and their lipid peroxidation and H2O2 levels are not affected by the salt treatment. The activities of all these enzymes remain at control level in peroxisomes of salt-treated Lem plants. The saltinduced increase in the antioxidant enzyme activities in the Lpa plants confer cross-tolerance toward enhanced mitochondrial and peroxisomal reactive oxygen species production imposed by salicylhydroxamic acid and 3amino-1,2,4-triazole, respectively (Mittova et al., 2003). 3.8. Effects of salinity on nitrogen metabolism Nitrate reductase activity (NRA) of leaves decreases in many plants under salt stress (AbdElBaki et al., 2000; Flores et al., 2000). The primary cause of a reduction of NRA in the leaves is a specific effect associated with the presence of Cl salts in the external medium. This effect of Cl seems to be due to a reduction in NO 3 uptake and consequently a lower NO 3 concentration in the leaves, although a direct effect of Clon the activity of the enzyme cannot be discarded (Cram, 1973; Smith, 1973; Deane-Drummond and Glauss, 1982; Flores et al., 2000). In Zea mays the nitrate content of leaves decreases, but it increases in roots under NaCl stress and NRA of leaves also decreases under salinity (AbdElBaki et al., 2000). Soussi et al. (1999) have

reported that salinity inhibits nitrogen fixation by reducing nodulation and nitrogenase activity in chickpea (C. arietinum L.). Considerable inhibition of nodulation and N2 fixation has also been reported by other workers (Bekki et al., 1987). Severe salt stress reduces the leg hemoglobinn content and nitrogenase activity in soybean root nodules (Comba et al., 1998). The exposure of nodulated roots of legumes such as soybean, common bean, and alfalfa to NaCl results in a rapid decrease in plant growth associated with a shortterm inhibition of both nodule growth and nitrogenase activity (Serraz et al., 1998). Khan et al. (1998) have reported that salinity inhibits nitrate content and uptake and NRA in leaves of maize plants. Nitrogenase activity measured with regard to acetylene reduction (C2H2) decreases in common bean by short-term exposure to salinity (Serraz et al., 2001). Decrease in NRA activity and in total nitrogen and nitrate uptake have been reported in leaves of B. parviflora (Parida and Das, 2004). NADP-specific isocitrate dehydrogenase (ICDH) is a key cytosolic enzyme that links C and N metabolism by supplying C skeletons for primary N assimilation in plants. The characterization of the transcript McICDH1 encoding an NADP-dependent isocitrate dehydrogenase (NADP-ICDH; EC 1.1.1.42) has been reported from the facultative halophyte M. crystallinum L., focusing on salt-dependent regulation of the enzyme (Popova et al., 2002). The activity of NADP-ICDH in plants adapted to high salinity increases in leaves and decreases in roots. By transcript analyses and Westerntype hybridizations, expression of Mc-ICDH1 is found to be stimulated in leaves in salt-adapted M. crystallinum. By immunocytological analyses, NADP-ICDH proteins are localized to most cell types with strongest expression in epidermal cells and in the vascular tissue. In leaves of salt-adapted plants, signal intensities increases in mesophyll cells. In contrast to Mc-ICDH1, the activity and transcript abundance of ferredoxindependent glutamate synthase (EC 1.4.7.1), which is the key enzyme of N assimilation and biosynthesis of amino acids, decreases in leaves in response to salt stress (Popova et al., 2002). 3.9. Effects of salinity on malate metabolism NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) is responsible for the reduction of oxaloacetate to malate in the chloroplasts of higher plants. In M. crystallinum, steady state transcript levels for chloroplast NADP-MDH decrease transiently in the leaves after salt stress and then increase to levels greater than two fold higher than levels in unstressed plants, whereas transcript levels in roots are extremely low and are unaffected by salt-stress treatment and the salt-stressinduced expression pattern of this enzyme suggests that


3.10. Effects of salinity on chloroplast ultrastructure Electron microscopy shows that the thylakoidal structure of the chloroplasts becomes disorganized, the number and size of plastoglobuli increases, and their starch content decreases in plants treated with NaCl (Hernandez et al., 1995, 1999). In the mesophyll of sweet potato leaves, thylakoid membranes of chloroplast are swollen and most are lost under severe salt stress (Mitsuya et al., 2000). In potato, salt stress reduces the numbers and depth of the grana stacks and causes a swelling of the thylakoid and starch grains become larger in the chloroplasts (Bruns and Hecht-Buchholz, 1990). In leaves of NaCl-treated plants of tomato the transmission electron microscopy shows that the chloroplasts are aggregated, the cell membranes are distorted and wrinkled, and there are no signs of grana or thylakoid structures in chloroplasts (Khavarinejad and Mostofi, 1998). Chloroplast ultrastructural changes under salt stress are apparent in Eucalyptus microcorys; these include the presence of large starch grains, the dilation of the thylakoid membranes, the near absence of grana, and the presence of enlarged mesophyll cells (Keiper et al., 1998). We have also reported a notable disorganization of the thylakoid structure of chloroplasts in leaves of B. parviflora by salt stress (Parida et al., 2003). 3.11. Mechanism of salinity effects on photosynthesis Plant growth is the result of integrated and regulated physiological processes. Physiological processes are affected by number of environmental factors and they determine the response of plants to stress. Limitation of plant growth by environmental factors cannot be assigned to a single physiological process. The dominant physiological process is photosynthesis. Plant growth as biomass production is a measure of net photosynthesis and, therefore, environmental stresses affecting growth also affect photosynthesis. Salt stress causes either shortor long-term effects on photosynthesis. The short-term effect occurs after a few hours or within 1 or 2 days of the onset of exposure and this response is important, as

there is complete cessation of carbon assimilation within hours. The long-term effect occurs after several days of exposure to salt and reduction in carbon assimilation is due to the salt accumulation in developing leaves (Munns and Termatt, 1986). Although there are reports of suppression of photosynthesis upon salt stress (Chaudhuri and Choudhuri, 1997; Soussi et al., 1998; AliDinar et al., 1999; Romeroaranda et al., 2001; Kao et al., 2001), there are also reports that photosynthesis is not slowed down by salinity and is even stimulated by low salt concentration (Rajesh et al., 1998; Kurban et al., 1999). In A. pseudoalhagi (a leguminous plant), the leaf CO2 assimilation rate increases at low salinity (50 mM NaCl) but is not affected significantly by 100 mM NaCl, while it is reduced to about 60% of the control in 200 mM NaCl. Similarly stomatal conductance is consistent with the CO2 assimilation rate regardless of the treatments, and intercellular CO2 concentration is lower in the NaCl-treated plants than in the control (Kurban et al., 1999). In mulberry, net CO2 assimilation rate (PN), stomatal conductance (gs), and transpiration rate (E) decline under salt stress, whereas intercellular CO2 concentration (Ci) increases (Agastian et al., 2000). In B. parviflora net CO2 PN increases at low salinity (mM) and decreases at high salinity (Fig. 6), whereas gs remains the same as in control at low salinity and decreases at high salinity (Fig. 7) (Parida et al., 2004a). NaCl stress decreases the chlorophyll content and net photosynthetic rate and increases the rate of respiration and CO2 compensation concentration and there are no significant changes in the carotenoid contents in leaves of alfalfa plants (Khavarinejad and Chaparzadeh, 1998). In A. lentiformis, net CO2 assimilation rate and the ratio of ribulose 18 16 14 PN (µmolm-2 s-1)

it may participate in the CO2 fixation pathway during CAM (Cushman, 1993). When Eucalyptus citridora plants are treated with NaCl, an increased Na+ level is observed in shoots 3 weeks after treatment, and at this stage of treatment the growth of plants is not reduced but the malate metabolism is modified. The malate content decreases in leaves while the specific activities of NAD and NADP-malic enzymes increase. The stimulation in enzyme activity is more pronounced for NADPmalic enzyme but, for both enzymes, enzyme activity diminishes as early as 5 weeks after treatment (DeAragao et al., 1997).

341

12 10 8 0 mM

6

100 mM

4

200 mM 2

400 mM

0 7

14

30

45

Duration of NaCl treatment (d) Fig. 6. Effect of NaCl treatment on photosynthetic rate (PN) in B. parviflora measured as a function of days of NaCl treatment. Values are mean7SE (from Parida et al., 2004a).


342

biphosphate carboxylase (Rubisco) activity to that of phosphoenol pyruvate carboxylase (PEPC) decrease under salinity because PEPC activity on a leaf area basis increases linearly with salinity, while Rubisco activity remains relatively constant (Zhu and Meinzer, 1999). In the cyanobacterium S. platensis salt stress inhibits the apparent quantum efficiency of photosynthesis and photosystem II (PSII) activity while stimulating photosystem I (PSI) activity and dark respiration significantly. Salt stress also results in a decrease in overall activity of the electron transport chain, which cannot be restored by diphenyl carbazide, an artificial electron donor to PSII (Lu and Vonshak, 1999). Experimental evidence shows that PSII mediated electron transport activity increases at low salinity (100 mM) and decreases at high salinity in B. parviflora (Table 8; Parida et al., 2003). A significant reduction in CO2 assimilation rate and stomatal conductance by salt treatment is registered in horsegram (Macrotyloma 800 700

gs (mmolm-2 s -1)

600 500 400 0 mM

300

100 mM 200 200 mM 100

400 mM

0 7

14

30

45

Duration of NaCl treatment (d) Fig. 7. Effect of NaCl treatment on stomatal conductance (gs) in B. parviflora measured as a function of days of NaCl treatment. Values are mean7SE (from Parida et al., 2004a).

uniflorum) and salinity inhibits the photosynthetic electron transport and the activity of enzymes of the Calvin cycle, ribulose-1,5-biphosphate carboxylase (E.C.4.1.1.39), ribulose-5-phosphate kinase (E.C.2.7.1.19), ribulose-5phosphate isomerase (E.C.5.3.16), and NADP-glyceraldehyde-3-phosphate dehydrogenase (E.C.1.2.13) (Reddy et al., 1992). In four cultivars of rice (Oryza sativa L.), gradual decreases in the activities of PSI, PSII, and chlorophyll fluorescence transients and emission at 688 nm are observed with increase in NaCl concentration and a drastic decrease in net photosynthetic rate is found (Tiwari et al., 1997). Mishra et al. (1991) have reported that in wheat (T. aesivum L.) salt stress has no direct effect on electron transport activity and F(v)/F(m) ratio, suggesting that the efficiency of the photochemistry of PSII is not affected and decrease in F(m) due to salt stress may influence reduction of Q(A). These results on fluorescence indicate that salt stress predisposes photoinhibition of plants and reduces their ability to recover from photoinhibition. Light stress and salt stress are major environmental factors that limit the efficiency of photosynthesis. However, Allakhverdiev et al. (2002) have reported that the effects of light and salt stress on PSII in the cyanobacterium Synecchocystis sp. PCC 6803 are completely different. Strong light induces photodamage to PS II, whereas salt stress inhibits the repair of photodamaged PSII and does not accelearate damage to PSII directly. The combination of light and salt stress appears to inactivate PSII very rapidly as a consequence of their synergistic effects. Radioactive labeling of cells reveals that salt stress inhibits the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein of PSII. Northern and Western blotting analyses by Allakhverdiev et al. (2002) demonstrate that salt stress inhibits the transcription and the translation of psbA genes, which encode D1 protein. DNA microarray analysis indicates that the light-induced expression of various genes is suppressed by salt stress. It has been suggested that salt stress inhibits the repair of PSII via

Table 8 Effects of NaCl (mM) on PSII-mediated electron transport activity (mmol mg chl–1 h1) in B. parviflora as monitored with regard to DCPIP photoreduction [NaCl] (mM) H2O-DCPIP

0 100 200 400

DPC-DCPIP

0 100 200 400

0 days 72.9772.5 72.977 2.7 72.9772.0 72.9772.3 113.4072.7 113.8972.0 112.5772.0 112.8071.5

15 days

30 days

45 days

71.1271.3 75.1071.5 70.5271.5 68.2772.0

71.8672.0 75.3571.3 65.6371.7 60.8271.0

70.6871.7 75.8171.5 61.2871.0 54.8172.5

111.6271.3 113.5171.8 110.7071.5 104.8272.0

111.5871.0 115.5472.3 106.7372.5 85.8172.4

110.5571.8 114.6272.5 93.4772.3 78.6571.7

The rate was measured at different days after NaCl treatment in a reaction mixture at pH 7.0 as H2O-DCPIP without any uncoupler and inhibitors or as DPC-DCPIP. The values are mean7SE (n ¼ 6) (Source: Parida et al., 2003).


suppression of the activities of the transcriptional and translational machinery (Allakhverdiev et al., 2002). Photosynthesis involves a long chain of mechanisms, enzymes, and intermediate products and is regulated by several external and internal factors. Photosynthetic efficiency depends on the sequence of metabolic events such as photochemical reactions, on the enzymes involved in carbon assimilation, on the structure of the photosynthetic apparatus, and on the transport of photosynthetic intermediates between subcellular compartments. Photosynthetic rate is lower in salt-treated plants, but the photosynthetic potential is not greatly affected when rates are expressed with regard to chlorophyll or leaf area. Decreases in photosynthetic rate are due to several factors: (1) dehydration of cell membranes which reduce their permeability to CO2, (2) salt toxicity, (3) reduction of CO2 supply because of hydroactive closure of stomata, (4) enhanced senescence induced by salinity, (5) changes of enzyme activity induced by changes in cytoplasmic structure, and (6) negative feedback by reduced sink activity (Iyengar and Reddy, 1996). Photosynthetic activity decreases as water potential of leaves decreases (Iyengar and Reddy, 1996). The reduction in photosynthetic activity depends on two aspects of salinization, i.e., the total concentration of salt and their ionic composition. High salt concentration in soil and water create high osmotic potential, which reduces the availability of water to plants. Decrease in water potential causes osmotic stress, which reversibly inactivates photosynthetic electron transport via shrinkage of intercellular space which is due to efflux of water through water channels in the plasma membrane (Allakhverdiev et al., 2000a). Increase in osmotic potential under high salt conditions causes Na+ ions to leak into the cytosol (Papageorgiou et al., 1998) and inactivate both photosynthetic and respiratory electron transport (Allakhverdiev et al., 1999). High salt (NaCl) uptake competes with the uptake of other nutrient ions, especially K+, leading to K+ deficiency (Ball et al., 1987). Under such conditions of high salinity and K+ deficiency, a reduction in quantum yield of oxygen evolution due to malfunctioning of photosystem II occurs (Ball et al., 1987). The reduction in photosynthetic rate is also due to the reduction in stomatal conductance resulting in restricted availability of CO2 for carboxylation reactions (Brugnoli and Bjorkman, 1992). Stomatal closure minimizes loss of water by transpiration and this affects chloroplast light-harvesting and energy-conversion systems thus leading to alteration in chloroplast activity (Iyengar and Reddy, 1996). The extent to which stomatal closure affects photosynthetic capacity depends on the magnitude of partial pressure of CO2 inside the leaf. There are also reports of nonstomatal inhibition of photosynthesis under salt stress. This nonstomatal inhibition is due to

343

increased resistance to CO2 diffusion in the liquid phase from the mesophyll wall to the site of CO2 reduction in the chloroplast and reduced efficiency of RUBPCase (Iyengar and Reddy, 1996). Reduction in photosynthetic capacity is also a consequence of inhibition of certain carbon metabolism processes by feedback from other salt-induced reactions (Greenway and Munns, 1980). Under reduced water potential, stromal levels of the substrate fructose-1,6bisphosphate (FBP) accumulate and the FBPase product fructose-6-phosphate is reduced so that FBPase becomes rate limiting to photosynthesis (Heuer, 1996). To cope with salt stress plants respond with physiological and biochemical changes that aim at the retention of water despite high external osmoticum and the maintenance of photosynthetic activity and these enable plants to become tolerant. An understanding of the mechanisms by which salinity affects photosynthesis would aid the improvement of growth conditions and crop yield and would provide useful tools for future genetic engineering.

4. Conclusion Salinity effects and problems with regard to tolerance and ecological performance are discussed briefly in this review. This review provides information on physiological, biochemical, and molecular bases of salt tolerance. Efforts have been made to compare the relative sensitivity of various plant species to salt, and uptake and transport of NaCl are considered with regard to phytotoxicity and their interactions with nutrients. Present knowledge offers some ways for increasing salt tolerance. In conclusion, salinity is the most serious threat to agriculture and to the environment in many parts of the world and key molecular factors that can be used for genetic engineering of salt-tolerant plants include overexpression of specific transcription factors, characterization of dehydrin proteins, overproduction of osmoprotectants, expression of water channel proteins and ion transporters, and expression and characterization of molecular chaperones.

Acknowledgments We are grateful to Professor P. Mohanty, Visiting Professor, Regional Plant Resource Centre, Bhubaneswar for his valuable suggestions during the course of studies. The financial assistance from CSIR (Grant No. 38(983)/EMR-II), New Delhi is gratefully acknowledged.



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