7

Current Concepts about Salinity and Salinity Tolerance in Plants

Askım Hediye Sekmen, Melike Bor, Filiz Ozdemir, and Ismail Turkan

Abstract

Soil salinity causes a significant reduction in plant yield and productivitydepending on the reduction of the influx of water into the roots caused by the highexternal osmotic potential in the soil. Due to salinity problems worldwide, eachyear about 2–3Mha of land go out of agricultural production. Therefore, thedevelopment of salinity-tolerant crops is crucial for sustainable agriculture. Hence,understanding the physiological, biochemical, and molecular basis of plant salttolerance will help to improve salt stress tolerance in plants. It has been reported bydifferent researchers that salt tolerance is a complex trait interacting with plantmetabolism, leading to inhibition of growth, development, and reproduction ofplants. Salt tolerance requires the involvement of several different traits, such asthe accumulation and compartmentalization of ions, the synthesis of compatiblesolutes for osmotic adjustment, the ability to accumulate essential nutrients suchas Kþ and Ca2þ in the presence of high concentrations of Naþ, the ability to limitthe entry of these saline ions into the transpiration stream, to continue to regulatetranspiration in the presence of high concentrations of Naþ and Cl�, and anefficient reactive oxygen species-scavenging capacity. In this chapter, salt stressperception by plants, the plant responses to salt stress, and the regulatorymechanism that allows plants to cope with stress are described, and informationfrom recent studies concerning salinity tolerance are discussed.

7.1

Introduction

Due to salinity problems worldwide, each year about 2–3Mha of land go out ofagricultural production. The impact is more prominent in arid and semi-arid areas,and the great proportion of the world’s food supply is produced in such areas byusing irrigation systems. High quantities of irrigation water and low quantities ofrainfall lead to irrigation-induced salinity, and rising saline groundwater levelscombined with inadequate leaching are the major reasons for soil salinity that

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Climate Change and Plant Abiotic Stress Tolerance, First Edition. Edited by Narendra Tuteja and Sarvajeet S. Gill.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

severely affects plants [1]. On the other hand, the causes of soil salinity can be dueto different reasons – sea water intrusion into aquifers in Mediterranean countriesand the deposition of salts carried by wind and rain in the Australian continent arewell-known examples [2]. In agricultural terms, salinity is characterized by theconcentration of soluble salts, NaCl in particular, in the soil, causing a significantreduction in the yield and productivity depending on the reduction of the influx ofwater into the roots caused by the high external osmotic potential in the soil [2,3].In addition to the discrepancies in salinity-withstanding threshold values of crops,the impact of salinity on crop plants may increase due to the effects of otherenvironmental factors under field conditions.

7.2

What is Salt Stress?

Usually soil salinity levels are defined by the electrical conductivity (ECe) values ofthe dissolved salt (NaCl is the major concern and most soluble one) in saturatedsoil conditions. Soils are classified as saline when the ECe value is 4 dSm�1 ormore [4]. This value approximates to 40mM NaCl and generates an osmoticpressure of almost 0.2MPa, which is significantly effective on crops such as rice,clover, corn, bean, and chickpea [5,6]. The demand for agricultural productivityincreases as the human population rises and crop production in stressfulenvironments, including saline soils, has become a major challenge. Salinity limitsthe yield of agricultural crops as the majority of crop plants are glycophytes andthey do not display any salt tolerance mechanisms [2]. There are prominentdiscrepancies between glycophytes and halophytes in terms of salinity survival andgrowth capacity as the latter have an evolutionary background for their morpholo-gical and phlyogenetic adaptations [7].Characterization and determination of candidate genes in order to develop salt-

tolerant crop species is the fundamental focus of salt stress research. Since salinitytolerance is a developmentally regulated polygenic and complex trait, it is difficultto isolate one or a few genes conferring increased salt tolerance in plants. Forinstance, using the microarray approach, Ma et al. [8] have identified over 1500genes in Arabidopsis thaliana that were strongly salt regulated and most of thesegenes have revealed interactions within complex stress signaling networks.Understanding the physiology of salt tolerance is one of the major concerns of

plant biology and to date several researchers have reported that salt tolerance is acomplex trait including several interactions within plant metabolism. Salt tolerancerequires the involvement of several different traits, such as the accumulation andcompartmentalization of ions, the synthesis of compatible solutes for osmoticadjustment, the ability to accumulate essential nutrients such as Kþ and Ca2þ inthe presence of high concentrations of Naþ, the ability to limit the entry of thesesaline ions into the transpiration stream, the ability to continue to regulatetranspiration in the presence of high concentrations of Naþ and Cl�, and efficientROS scavenging capacity [7,9]. For plants living in saline environments (halophyte

164 7 Current Concepts about Salinity and Salinity Tolerance in Plants

plants), the Naþ concentration can range from around 100 to 2380mM Na, whichis equivalent to a water potential range from �0.5 to �11.0MPa [7]. Interesting anddiverse adaptive mechanisms for such salt levels have been evolved in these plants;one good example is the high Naþ and Cl� exclusion capacity of sea barley grass(Hordeum marinum) at 450mM NaCl [10], which is deleterious for the crop species.The effects of salinity on plants differ according to the genotype, developmental

stage, age, and plant organ. Moreover, the extent of these effects is dependent onthe type, period, and composition of the salinity. Munns [11] indicated thatresponses of plants to salinity at different time scales showed variations. However,the typical response seen both in sensitive and tolerant plants is the reduction ofleaf growth and emergence rates, which are more prominent as compared to thoseof the root. Since a high Naþ concentration is toxic for all plant cells, maintenanceof a high Kþ/Naþ ratio is essential for normal growth and development in mostplant species. In roots and shoots, nutritional imbalances such as increased Naþ

with decreased Kþ and Ca2þ levels affect growth in relation to reduced cell divisionand elongation [12].Whole-plant performance under salt stress conditions is the key component of

the definition of salinity tolerance in plants. There are several descriptionsregarding salinity tolerance in plants as emphasized by Cimato et al. [13]; control ofsalt entry and allocation of the salt at organism level is defined as the “physiologicaltolerance,” and the ability to maintain biomass production and crop yield isreferred as “horticultural tolerance” [14,15]. On the other hand, the concept ofsalinity tolerance also differs between annual crops and long-lived woody species.For instance, in Mediterranean evergreen plants such as Olea europaea, survival ismore important than growth performance since these plants are exposed to highsoil salinity levels during 2–3 years of a leaf’s lifespan [11,13,15]. Nevertheless, inannual crops such as wheat and rice, tolerance is considered as the maintenance ofa relatively better growth rate under salt stress conditions [13,14,16].Different threshold values were defined by scientists for discriminating

halophytes from glycophytes. Aronson [17] classified halophytes as specieswithstanding salt concentrations over 80mM NaCl (7.8 dSm�1), but Flowers andColmer [9] stated this limit was too low for salt-tolerant plants and set a newthreshold value of 200mM NaCl. According to Flower and Colmer [9], halophytesthat can tolerate such salt levels are not more than 0.25% of the knownangiosperms, and list 350 species including succulents and saltbushes distributedamong different orders, such as Caryophyllales, Alismatales, Malpighiales, Poales,and Lamiales [7].Halophytes occupy the native flora of saline environments, and they have the

capacity to accommodate extreme salinity with special anatomical and morphologi-cal adaptations or avoidance mechanisms [18]. These adaptations and mechanismsall contribute to their ability to cope with salinity for survival and better growth.Early vacuolation and promotion of suberization of the hypodermis and endoder-mis with the well-developed Casparian strip in roots of halophytes (Suaedamaritima) and salt glands on the leaves of mangrove species (Avicennia marina) forsalt extrusion are well-known characteristics of salt-tolerant plants [19].

7.2 What is Salt Stress? 165

Researchers are more focused on understanding the mechanisms of Naþ

exclusion and control of its transport within the plants, as Naþ reaches toxic levelsbefore Cl� under salt stress conditions [6]. It is believed that the regulation of genesencoding specific ion channels and transporters is important for both Naþ

transport and exclusion, and elucidation of these gene expression patterns is themajor target for improving new molecular breeding strategies. The ability tocompartmentalize the ions in vacuoles and discriminate in favor of Kþ over Naþ isthe main characteristic of salt tolerance. Halophytes that accumulate Naþ and Cl�

at sufficient concentrations for osmotic adjustment and avoid toxic levels of theseions at the transpiration stream use these traits better than glycophytes.Although salt-tolerant halophytes and salt-sensitive glycophytes have common

genes for stress responses in their genomes [20], better regulation and expressionof these genes in halophytes than in glycophytes is due to differential regulationand expression of these genes within these two groups. Confirmation of this comesfrom the work of Taji et al. [21] who compared microarray profiles of the salt-tolerant halophyte Thellugiella halophila and its close relative A. thaliana. Undersalinity conditions, 40 genes in A. thaliana and only six genes in T. halophila wereupregulated. However, under control conditions, a large number of stress-relatedgenes were expressed at high levels in the latter [21,22]. One good example is theincreased expression of an AtSOS1 homolog in the plasma membrane resulting inincreased Hþ transport and hydrolytic activity of the Hþ-ATPase in T. halophila [23].Studies mentioned above paved the way for the identification and cloning of suchgenes in different halophytic plants, such as two AtSOS1 homologs in Chenopo-dium quinoa [24] and an AlNHX (AtNHX homolog) in a monocot halophyte,Aeluropus littoralis [25]. It was shown that these cloned genes conferred salttolerance. In a recent study, when clover (Medicago sativa) was transformed bySsNHX1 from Salsola soda, it showed increased tolerance to high NaCl salinity [26].Moreover, secreting excess ions through glands, synthesizing organic com-

pounds in the cytoplasm for osmotic adjustment, and recirculation of ions fromshoots to roots are some of the other tolerance mechanisms found in halophytespecies [6,7,9]. According to Zhu [27], ion contents differ in the two plant groups;halophytes accumulate and glycophytes exclude salts under high salinity. Halo-phytes such as Salvadora persica, Batis maritima, Spartina alterniflora, and Salicorniabigelovii can accumulate high NaCl levels with minimum growth reduction [28,29].Among the glycophytes, beans and maize are good examples of Naþ excluders,and bread wheat is also capable of keeping low rates of Naþ transport to the shoots[30–32].The widespread occurrence of halophytes among the higher plants indicates

their polyphyletic origin and it is not surprising to see variations in tolerance toextreme salinity levels. For instance, among different halophytic plants, whileAtriplex vesicara gives a high yield in the presence of 700mmol l�1 NaCl, Salicorniaeuropaea remains alive at 1020mmol l�1 NaCl [27].The ability of halophytic plants to grow under saline conditions evoked an

approach for “biosaline agriculture,” which is defined as the agricultural practicesperformed under different ranges of salinity in groundwater and soils [33]. In a

166 7 Current Concepts about Salinity and Salinity Tolerance in Plants

review by Lal [34], the biosaline agriculture concept for sustainable land use inharsh eco-regions and in biofuel production was emphasized. Halophytes irrigatedwith saline (brackish) or sea water can produce high biomass that has longerresidence periods because of slow decomposition rates. There were different rolesattributed to halophytes, such as biofuel feedstock, biomass for high-grade oil, andreclaiming salt-affected soils. There are different studies showing that plants suchas puccinellia (Puccinnelia stricta), tall wheatgrass (Thinopyrum ponticum), balansaclover (Trifolium michelianum), Italian ryegrass (Lolium multiflorum), salt watercouch (Paspalum vaginatum), and sweet clover (Melilotus alba) have a high dry massproduction capacity ranging between 4 and 10 t drymass ha�1 year�1 [33]. Oneextreme example is the high growth and biomass capacity of saltbush (Atriplexspp.), which produces 2.2–5.3 t drymass ha�1 year�1 when irrigated with salinedrainage water [35]. Salt-tolerant plants can also be used in formulated feeding(e.g., Salicorniameal has been used for maize and soybean meal in broiler diets [36]and in fish diets [37]). However, the extent of utilization depends on yield of theedible biomass and costs of harvesting and processing.

7.2.1

Perception of Salt Stress --- Still a Mystery

Direct or indirect perception mechanisms could be employed by the cell in orderto sense environmental stresses; as salinity has osmotic and ionic components,different perception hypothesis were proposed [38]. Wood et al. [39] defined aputative direct osmosensor that would act as a ligand-specific receptor fordetecting water activity and indirect perception mechanisms that were affectedby osmotic changes, including cell volume, turgor pressure, membrane stability,individual solute concentrations, ionic strength, and accumulation of macro-molecules in the cytoplasm. Histidine kinases are among the best candidates assalt and osmotic stress receptors that have been defined as osmosensors inprokaryotes and yeast [38,40–42]. Histidine kinase 1 (AtHK1) has been proposedas an osmosensor in A. thaliana, which is also involved in the regulation of thedesiccation process during seed maturation [43–45,72]. Gene expression andphenotypic analysis of mutant plants have provided evidence supporting theroles of histidine kinases as sensors for salt and osmotic stress; however, to datethe direct evidence underpinning the mechanisms of this stress perception isnot clear [38].Although the effects of salt stress and responses of different plants have been

extensively studied and well known, the perception of Naþ is still a mystery. We stilldo not know whether it is sensed outside or inside the plant cell. It was proposedthat Naþ outside the cell can be sensed by a specific membrane receptor and Naþ inthe cytoplasm can be perceived by Na-sensitive enzymes or membrane proteins [2].However, a plasma membrane Naþ/Hþ antiporter that is encoded by salt overlysensitive 1 (SOS1) may be one of these sensors as defined by Zhu [20]. SOS2protein kinase, a member of the sucrose non-fermenting 1-related protein familyregulates SOS1, which is regulated by the Ca2þ-sensing protein SOS3 [20].

7.2 What is Salt Stress? 167

Screening of salt-hypersensitive mutants provided detailed information on themembers of the SOS pathway. SOS4 encodes a pyridoxal kinase that is involved inthe biosynthesis of pyridoxal-5-phosphate (PLP) [46]. SOS5 is shown to be aputative cell surface [47] adhesion protein, which is a good candidate to be a Naþ

sensor [48,49]. The Ca-mediated SOS pathway is well characterized in A. thaliana,and controls ion homeostasis and salinity tolerance in a hierarchical way [20,50,51].More information about SOS genes and SOS signaling pathway in relation tosalinity tolerance will be given and discussed later in this chapter. The perception ofosmotic effects of salinity is proposed to be related to the activation of stretch-activated channels, redox mediated-systems, and transmembrane kinases.

7.2.2

Salt Stress Signaling: Now, We Know Better

After the perception of salt stress by the putative receptors, the generation ofsecond messengers such as Ca2þ, pH, reactive oxygen species (ROS), and inositolphosphates (IP3) induces the switch on the stress-responsive genes for mediatingstress tolerance [46].

7.2.2.1 Ca2þ Signaling

Calcium is an essential nutrient for growth and development of plants [52].Under normal conditions, in plant cells, the cytosolic concentration of calcium[Ca2þ]cyt is mostly 10–200 nM, whereas its concentration in the cell wall,vacuole, endoplasmic reticulum, and mitochondria varies between 1 and 10mM[53,54]. After the perception of salt stress, although a sudden increase in[Ca2þ]cyt was recorded in many studies [50,55–59], there are also studiesshowing a reduction in [Ca2þ]cyt levels in [60,61]. Within minutes of applicationof 100mM NaCl to root cells of A. thaliana [60,61] or to corn root protoplast[62], a decrease in [Ca2þ]cyt was observed. These results showed that a change inthe cytosolic free Ca2þ concentration is one of the earliest intracellular reactionsto abiotic stress signals [63]. These fast and transient changes in theconcentration of free Ca2þ ions, which are called “Ca2þ signals,” trigger a wholerange of signal transduction pathways via different calcium sensors thatare either induced or are already present in the cell [64,65]. These signals aredecoded by different calcium sensor proteins including calmodulins (CAMs),calcineurin B-like proteins (CBLs), protein kinase effectors including Ca2þ-dependent protein kinases (CDPKs), CDPK-related kinases (CRKs), calmodulin-dependent protein kinases (CaMKs), CBL-interacting protein kinases (CIPKs,also called SnRK3s), and Ca2þ- and calmodulin-dependent protein kinases(CCaMKs) [66]. Among these, CDPKs and CBL/CIPK pairs are able to translatethe transient change in the [Ca2þ]cyt concentration into a phosphorylationsignal. In addition, other protein kinases such as mitogen-activated proteinkinases (MAPKs: a MAPK kinase kinase (MKKK), a MAPK kinase (MKK), and aMAPK) or Snf1-related kinases (SnRKs) become activated in response to thesame stimuli as well [66–68]. In early studies, a major function of MAPKs was

168 7 Current Concepts about Salinity and Salinity Tolerance in Plants

found to be to translate an extracellular stimulus into an appropriate cellularresponse via transcriptional induction of stress-responsive genes or directregulation of enzymatic activities or channel proteins in the immediateresponse [69]. Recently, both CDPKs and MAPKs have been implicated incross-tolerance between biotic and abiotic stress responses. For example,Abuqamar et al. [70] observed an increase in salt tolerance of tomato bywounding or overexpression of pathogen-induced MYB transcription factors(R2R3-MYB) and Capiati et al. [71] reported the interaction of CDPK in this typeof cross-tolerance. Moreover, several studies showed that many of these CDPKsand MAPKs present differential patterns of expression under various abioticstress conditions [64]. The functions of some of CDPKs and MAPKs inA. thaliana and Oryza sativa under salt stress were given in Table 7.1.

7.2.2.2 pH in Stress Signaling

In addition to Ca2þ, protons also function as secondary messengers in response todifferent stress conditions, including salinity stress. In plant cells, under normalconditions, cell cytosol pH (pHcyt) is around 7.5, while the apoplast and vacuolarlumen pH are around 5.5 [92]. However, Gao et al. [93] reported that ionic stresscaused a decline in pHcyt, whereas osmotic stress did not alter pHcyt in Arabidopsisroots. Similarly, Kader et al. [57] showed changes in pHcyt during plant defenseresponses against salt stress in rice. In the same study, ionic stress induced atransient cytosolic acidification in salt-sensitive rice under salt stress. These resultsshowed that (i) increased [Ca2þ]cyt under salinity stress was attributed to the ioniccomponent of salinity stress, not the osmotic stress [57,94], and (ii) pHcyt is relatedto a change in vacuole pH depending upon Hþ movement occurring between thecytosol and vacuole [52].

7.2.2.3 Abscisic Acid Signaling

Limited water supply as a result of salt stress leads to an immediate hydraulicsignal in plants that triggers abscisic acid (ABA) biosynthesis over long distances[95]. After the perception of this signal, ABA produced in dehydrated roots istransported to the xylem [96,97]. Actually, Zhang and Davies [98] showed that xylemABA comes from two sources: from roots in drying soil (root-sourced ABA) andfrom older leaves that wilted earlier than the younger leaves (leave-sourced ABA).ABA concentration in the xylem rather than its flux regulates stomatal movementto reduce the water loss in the leaves. This mechanism is modified by the xylem/apoplastic pH and ionic conditions [99–101]. Drought, salinity, and high lightmight lead to an increase in xylem sap pH, which decreases removal of ABA fromxylem and leaf apoplast to symplast, allowing more ABA to reach the guard cell[102,103].The regulation of ion channels by ABA in stomata has been an interesting

research field for many years. Recent works have shown that ABA and Ca2þ

signaling pathways regulate both anion (SLAC1) and cation channels (KAT1), andvice versa [104]. While Ca2þ-dependent regulation is probably provided by CPK23,CPK3, and CPK6, which all stimulate SLAC1, ABA-dependent regulation in ion

7.2 What is Salt Stress? 169

channels provided by ABA-activated protein kinase OST1 (open stomata 1) andSnRKs [79,105]. In guard cells, these protein kinases regulate the key targets(SLAC1 and KAT1) of the ABA signaling pathway. Another key target of OST1,which activates and inhibits SLAC1 and KAT1, respectively, is a NADPH oxidase(NOX) that generates H2O2 [106]. OST1-dependent H2O2 production inactivatesABA coreceptors ABI1 and the related PP2CA, which inhibit OST1 and OST1-dependent SLAC1 activation [105,107–110].

Table 7.1 Functions of selected A. thaliana and O. sativa CDPKs and MAPKs under salt stress.

Species Protein Biological function under salt stress Reference

Arabidopsisthaliana

AtCDPK1, 2 Induced by salt [72]AtCPK3 Salt stress acclimation [73,74]AtCPK10, 11 Induction in response to salt stress [72]AtCPK21, 23 Negative regulator of salt stress sig-

naling[63,75]

AtCPK32 Hypersensitive response to salt stress [64]AtCIPK3, 9 Inhibition in response to salt stress [76]AtCIPK1 Mediates salt signaling pathway [77]AtCIPK6 Promoted salt tolerance [78,79]AtCIPK16 Upregulation at its transcript level

under salt stress[80]

AtMKK1 Not activated by salt stress [81]AtMKK2 A key signal transducer for salt stress [81]AtMKK9 Increases salt sensitivity [82]AtMEKK1 (a MAPKKK) Upregulation under high salt

condition[83]

An upstream activator of MKK2 [81]The downstream MAPKs MPK4 andMPK6

[81]

AtMKP1 Negative role in salt stress signalingthrough MAPKs (MPK6 and MPK4)

[84]

AtMEKK1, AtMPK3 Induction via salt stress [83]AtMPK1, AtMPK4, 6 Activation by salt stress [85]AtMAPKK, MKK2 Mediates salt stress signaling [81]

OryzaSativa

OsCDPK7 Induced by salt [86,87]OsCPK7, 13, 15, 20, 21 Upregulation under salt stress [88]OsCPK1, 17 Downregulation under salt stress [88]OsCIPK7, 8, 9, 10, 11, 15,16, 17, 21, 22, 23, 24, 29

Salt inducible [88]

OsCPK13 Induction in response to salt [88]OsCIPK3 Regulates an ABA-independent salt

stress[89]

OsMAPKK4, 6 Activated by salt stress [90]OsMAPKK1 Increased tolerance [90]OsMAPK5 ABA-inducible kinase, positively reg-

ulates tolerance to salt stress[91]

170 7 Current Concepts about Salinity and Salinity Tolerance in Plants

7.2.2.4 Phospholipid Signaling

Phospholipases catalyze the hydrolysis of the phosphodiester bond between thephosphate and the polar group (inositol, serine, glycerol, choline, and ethanola-mine). In plants, phospholipases are required for signal transduction in a-amylasesynthesis in aleurone cells, stomatal closure, pathogen responses, leaf senescence,auxin-stimulated cell elongation, oil biosynthesis during embryo maturation,membrane reorganization, and light-mediated processes [111–114]. In signaltransduction, IP3 (inositol 1,4,5-trisphosphate), diacylglycerol (DAG), and phos-phatidic acid, which are produced by the action of phospholipases, play roles assignal molecules. Moreover, the activity of phospholipases is regulated by [Ca2þ]cytsignaling cascades through a Ca2þ/phospholipid binding site [115]. Twelvephospholipases genes, which are classified into six types, were identified inArabidopsis genome: PLDa (3), b (2),c (3), d, e, and f (2) [116].

Phosphatidic Acid The phosphatidic acid level increases within minutes understress conditions, such as osmotic stress, oxidative stress, drought, pathogenelicitors, and ABA, while its levels are very low under normal conditions [117].Phosphatidic acid is generated via two distinct phospholipase pathways: (i)phospholipase D (PLD), which hydrolyzes structural phospholipids, and (ii) actionof phospholipase C (PLC) and DAG kinase [113]. Phosphatidic acid formation iscontrolled by interaction of Ca2þ, G-proteins, protein kinases, MAPKs, and ROS. Itis broken down by diacylglycerol pyrophosphate phosphatase [118]. Several PLDsare involved in salt-induced phosphatidic acid formation directly by hydrolyzingstructural phospholipids such as phosphatidylcholine [119]. Hong et al. [120] foundthat PLDa3-overexpressing seeds displayed more resistance to salt stress. However,in the same study, PLDa3-KO mutant plda3-1 seeds were e more susceptible to saltstress. Similarly, Hong et al. [121] also reported that plda1 pldd double mutants arenot responsible for the phosphatidic acid produced under salt stress. PLDeenhances growth under hyperosmotic stress imposed by high salinity and waterdeficiency [121]. In addition, Tuteja and Sopory [114] found that DAG kinase isdominant producer of phosphatidic acid.

IP3 Other second messengers in signal transduction are IP3 and DAG that canactivate protein kinase C and trigger Ca2þ release, respectively. Inositol polypho-sphate 1-phosphatase, which catabolizes IP3, is encoded by the FRY1 gene(Fiery 1). Additionally, Xiong et al. [122] found that fry1 mutants exhibited anenhanced induction of stress-responsive genes, ABA, cold, and salt stressesindicating that FRY1 locus negatively regulates IP3 levels and stress signaling[123]. Most studies reported that rapid and transient increases in IP3concentration were observed in plant tissues or cultured cells in response tosalinity and hyperosmotic stress [124–126]. For example, Drobak and Watkins[125] found that salts and osmotic agents increased levels of IP3 15-fold as oneof the initial responses. An increase in the concentration of IP3 in Arabidopsisplants under stress triggers signal transduction pathways together with changesin cytosolic Ca2þ levels as reported by DeWald et al. [127].

7.2 What is Salt Stress? 171

Diacylglycerol (DAG) DAG and IP3 are generated by phospholipid phosphatidyli-nositol 4,5-bisphosphate, which is hydrolyzed by PLC [48]. IP3 diffuses into thecytosol, while DAG remains in the membrane [114]. Their formation results inincreased levels of Ca2þ under salt stress, which triggers signal transductionpathways via different calcium sensors including CAMs and CBLs [48].

7.3

Effects: Primary and Secondary

Root cells uptake Naþ ions via voltage-dependent channels such as Kþ

transporter HKT1 and/or by Naþ leakage. It is well known that the first responseof plants to salt stress is related to the osmotic stress component, which is thenfollowed by the ion toxicity component, leading to severe effects due to thedisplacement of Kþ ions with Naþ and Cl� ions within the cell [128]. In short-term salinity, growth inhibition in root and leaf tissues is entirely due to thesudden changes in cell water relations as plants are exposed to NaCl or otherosmotica, indicating that the responses are not salt-specific [11]. The expansionrate of growing leaves and emergence of new leaves are reduced due to thedeclined leaf turgor in the first phase of salt stress. Upon long-term exposure tosalinity, important cellular processes such as protein stability, tRNA binding toribosomes, and some enzymatic reactions are affected, due to competitionbetween Naþ and Kþ ions [129]. In addition to the competition between Naþ andKþ for uptake, the increased concentration of Naþ ions in the soil reduces theactivity and availability of many essential nutrients. Hence, maintenance of ahigh intracellular Kþ/Naþ ratio is the key determinant of plant growth andsurvival under salt stress conditions.

7.3.1

Salt Primary Effects: Osmotic and Ionic Phases

Under salt stress, plant growth, development, and productivity are limited by theosmotic effect of the salt in the soil or the toxic effect of the salt within the plant.Salinity causes osmotic stress due to limited water absorption and ionic stress dueto accumulation of high levels of Naþ and Cl� ions, leading to intercellular toxicityand/or imbalance [16,50]. While the effect of the osmotic stress is seen as a rapidinhibition of growth rate of young leaves, the effect of ion-specific toxicity isrecognized as the increase in the rate of senescence in older leaves.When soil water deficit is sensed by roots, chemical signals, referred to as “root

signals,” are transmitted from roots to the shoot [130]. Munns and Cramer [131]found that ABA, which is found in xylem sap, is the evident candidate for thissignal. However, recent studies showed that the chemical signal coming from theroots is not only ABA, but also other hormones such as cytokinins [132,133].Hormonal regulation during this osmotic phase in both roots and shoots is criticalto delay the accumulation of ions to toxic levels [132].

172 7 Current Concepts about Salinity and Salinity Tolerance in Plants

The osmotic effect of the salt around the roots leads rapidly to loss of cell volume,turgor, and growth inhibition of leaves. Moreover, the leaf area of the salt-treatedplants decreases, but the thickening of the leaves indicates that cell size and shapehave changed [134]. However, within hours, cells regain their original volume,although cell elongation continues to reduce [135–137].Root growth is usually less affected than shoot growth. Root architecture is

affected when salinity is perceived; degree of branching or rate of branchelongation changes and root elongation rate decreases [138–141]. Root elonga-tion rate can recover remarkably well after exposure to NaCl or other osmotica[11]. Hummel et al. [142] showed that the proportion of Arabidopsis roots canincrease in drying soil. Similarly, Lovisolo et al. [143] also found that grapevineroots continue to grow into deeper layers of soil. Continuation of root growthunder salt stress may provide additional surfaces for sequestration of toxic ions,leading to a lower salt concentration around roots [144]. On the other hand,Wu et al. [145] found that expansins, which are a family of plant proteinsessential for acid-induced cell wall loosening [146], are closely correlated withroot elongation. Additionally, adaptive wall loosening and growth maintenancein the apical region of maize roots are partly due to altered expansin geneexpression at low water potentials.Ionic stress has less effect on growth rates at especially moderate salinity, as

compared to osmotic stress [6]. However, at high salinity levels or in salt-sensitivespecies, the ionic effect overwhelms the osmotic effect. While dicot halophytesshow optimal growth in 100–250mM NaCl [9], the optima are much lower formonocot species [147].Sodium is toxic to many organisms, except for halotolerant organisms like

halobacteria and halophyte plants, which have specific mechanisms that keepintracellular Naþ concentrations low [144]. However, Storey and Walker [148] foundthat Cl� is considered to be the more toxic ion for some species such as citrus. Thisstatement does not imply that Cl� is more metabolically toxic than Naþ, ratherthese species are better at excluding Naþ from the leaf blades than Cl�.High NaCl concentrations (above 400mM) inhibit most enzymes involved in

carbohydrate metabolism via perturbation of the hydrophobic/electrostatic balancebetween the forces maintaining protein structure [144]. However, some enzymesare sensitive to lower Naþ concentration [6,149].Plant cells may overcome salt stress by (i) compartmentalizing salt in the

plant vacuole, (ii) extruding Naþ to the external medium or to the apoplast, and(iii) restricting Naþ permeation.In plants, the central vacuole plays a vital role in regulation of cytoplasmic ion

homeostasis [144]. Vacuolar compartmentalization of Naþ is achieved via theexpression and activity of tonoplast Naþ/Hþ antiporters, which use the protonmotive force generated by the vacuolar Hþ-translocating enzymes, Hþ-ATPase andHþ-inorganic pyrophosphatase (PPiase) [150].Improved salt tolerance in transgenic plants was found in either transformed or

overexpressed AtNHX1 and vacuolar Naþ/Hþ antiporters such as rice [151], tallfescue [152], and cotton [153].

7.3 Effects: Primary and Secondary 173

The transformation of AtNHX1 and vacuolar Naþ/Hþ antiporter from Arabidop-sis improved salt tolerance of Brassica napus [154], tomato [155], cotton [153], wheat[156], beet [157], and tall fescue [152]. Transformation of another Naþ/Hþ

antiporter family member, AtNHX3, in sugar beet (Beta vulgaris L.) resulted inincreased salt accumulation in leaves, but not in the roots, with enhancedconstituent soluble sugar contents [158]. Tonoplast Naþ/Hþ antiport activity wasinduced by NaCl treatment in roots of salt-tolerant Plantago maritima, but not insalt-sensitive Plantago media [159].Salinity leads to an increase in different ATPase activities in halophytes,

whereas they remain constant or decline in glycophytes [115]. Vera-Estrella et al.[23] reported increases in Hþ transport and H-ATPase hydrolytic activity in thetonoplast of NaCl-treated Thellungiella root cells. Similarly, Li et al. [160] alsoshowed overexpression of the Arabidopsis H-PPiase under salt stress. However,downregulation of V-ATPase expression in Mesembryanthemum roots under saltstress was reported by Golldack and Dietz [161]. V-ATPase activities in roots ofP. maritima and P. media were shown to be unchanged by 50mM NaCl stressby Staal et al. [159]. These results suggest that vacuolar sequestration of Naþ inroots might not be a key determinant of salinity tolerance in halophytes asreported by Munns [130].Members of the HKT gene family functioning as Naþ/Kþ symporters and as

Naþ-selective transporters mediate the removal of sodium from the xylem, whichreduces the rate of sodium transfer to the shoot tissue [162]. It was reported thatAtHKT1 mutants are salt-sensitive as compared to wild-type. They hyperaccumu-late Naþ in the shoot, but show reduced accumulation of Naþ in the root [163,164].Moreover, Rus et al. [165] showed that mutation in AtHKT1 suppresses thehypersensitivity of sos3 mutants, suggesting that the wild-type SOS3 may inhibitthe activity of AtHKT1 [165].Naþ exclusion from leaves is associated with salt tolerance in cereal crops,

including rice [166,167], durum wheat [168], bread wheat [169], barley [170], wildrelatives ofHordeum species [10], and tall wheatgrass [171].Overall findings suggest that a high degree of exclusion of Naþ and Cl� from the

leaves is achieved by (i) tightly controlled uptake from the soil [172] and (ii)regulated movement in the xylem, by controlled loading of Cl� into the xylem [173]or by retrieval of Naþ as it moves in the transpiration stream to the leaves [174].

7.3.1.1 Role of the SOS Pathway in Ion Homeostasis

For regulation of ion transport under salt stress, a signaling pathway based on theSOS genes has been established, mainly including SOS1 (a plasma membraneNaþ/Hþ antiporter), SOS2 (a Ser/Thr protein kinase), and SOS3 (a myristoylatedCa-binding protein). SOS1 promotes efflux of excess Naþ ions to prevent plantsfrom the harm of Naþ accumulation. The SOS1 exchanger is phosphorylated andenhanced by the SOS2/SOS3 kinase complex mediated through a salt-inducedCa2þ signal (Figure 7.1). SOS1 plays a critical role in the salt resistance of higherplants. Oh et al. [175] found that the downregulation of ThSOS1 (Thellungiellahalophila SOS1) converted this halophyte into a salt-sensitive plant. Similarly, Xiong

174 7 Current Concepts about Salinity and Salinity Tolerance in Plants

and Zhu [176] and Zhu [177] reported that Arabidopsis sos1 and sos2 mutants arehypersensitive to NaCl due to their intracellular Naþ imbalance. Moreover, inthe same study, it was found that sos1 and sos2 mutants displayed microtubuledisruption. Furthermore, Wang et al. [178] also indicated the important role ofthe SOS pathway in the organization and dynamics of the cytoskeleton andmicrotubule network, which are also crucial in response to salt stress [178].In Arabidopsis, SOS2 is activated by the Ca2þ-binding protein SOS3 and

physically interacts with SOS3 [179–181]. The regulatory domain of SOS2 containsFISL and PPI motifs. While the FISL motif binds to SOS3, the PPI motif binds tothe type 2C protein phosphatase ABI2 [179]. SOS2 (together with SOS3) enhancesboth activities of Naþ/Hþ antiporter SOS1 and tonoplast NHX, which providesodium transport into the apoplast and vacuole, respectively, in response to saltstress. Deletion of SOS1 affects proton flux, resulting in changes in the protongradient, cytoplasmic pH [182], and activity of vacuolar Ca2þ/Hþ transporters(CAX1 and CAX2) [183]. Under salt stress, increasing cytosolic [Ca2þ], whichinitiates stress signal transduction, is perceived by SOS3. The activated SOS2/3complex can trigger CAX1 [184], NHX1, or other transporters involved in vacuolarNaþ transport [185]. On the other hand, Quan et al. [186] reported SOS3-likecalcium-binding protein 8 (SCaBP8), which along with SOS3, is required for theactivation of SOS2. Lin et al. [187] reported that SOS2 also phosphorylates andactivates downstream SCaBP8, but not SOS3.The SOS4 gene, which encodes the vitamin B6 salvage pathway enzyme

pyridoxal kinase, was firstly characterized by Shi et al. [188]. This gene is requiredfor biosynthesis of PLP and root hair development in Arabidopsis. In the samestudy, SOS4 was defined as a novel regulatory determinant of Na and Khomeostasis in plants. Gonz�alez et al. [189] demonstrated that the Arabidopsis sos4mutant substantially accumulates PL (pyridoxal), PN (pyridoxine) and PM(pyridoxamine) – a feature that was attributed to an upregulation of the de novobiosynthesis of the vitamin in this mutant. This mutant is sensitive to salt andosmotic stress, but exhibits increased drought tolerance [189] (Figure 7.1).SOS5 was shown to be a putative cell surface [47] adhesion protein, which is a

good candidate to be a Naþ sensor as it resides in the outer surface of the plasmamembrane with arabino-galactan protein-like and fasciclin-like domains [48,49].SOS6, which encodes a cellulose synthase-like protein, AtCSLD5, has been

recently characterized by Zhu et al. [190]. This protein is required for osmotic stresstolerance in Arabidopsis. Ebine et al. [191] reported that the atcsld5 mutant exhibitsphenotypes similar to those of the ara6 mutant. ARA6 mediates a traffickingpathway from endosomes to the plasma membrane. It was found that over-expression of constitutively active ARA6 elevated resistance to salinity stress.Recently, Fan et al. [192] found a new protein kinase gene SSG1 (salt sensitive

during seed germination 1). ssg1-1 and ssg1-2 mutants are hypersensitive to Naþ andosmotic stress. In these plants, transcript expression levels of the four SOS genes(SOS1, SOS2, SOS3, SOS4) and two transporters (AtNHX, AtNKT) were alldownregulated, indicating SSG1 can be an upstream component in the SOSsignaling pathway.

7.3 Effects: Primary and Secondary 175

7.3.2

Salt Secondary Effect: Oxidative Stress

Salt stress, like other environmental stresses, induces the accumulation of ROSsuch as hydrogen peroxide (H2O2), superoxide (O2

��), and hydroxyl radicals(OH�). ROS are produced in aerobic metabolism as byproducts of differentmetabolic pathways such as mitochondrial and chloroplast electron transport, andoxidation of glycolate (photorespiration), xanthine, and glucose. Although lowlevels of ROS may act as a signaling molecules in order to trigger defense genes,excess accumulation of ROS can destroy the cellular redox homeostasis leading tooxidative damage to membrane lipids, proteins, and nucleic acids [193]. To avoidthe accumulation of these compounds to toxic levels, plants possess a complexantioxidant defense system including non-enzymatic antioxidants, such as ascorbicacid, glutathione, tocopherols, and carotenoids, and enzymatic antioxidants, suchas superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6),peroxidase (POX; EC 1.11.1.7), and ascorbate peroxidase (APX; EC 1.11.1.11)enzymes [194].

Figure 7.1 SOS signaling pathway for salt stress adaptation in higher plants.

176 7 Current Concepts about Salinity and Salinity Tolerance in Plants

SOD converts O2� to H2O2, which is detoxified to water and oxygen by CAT, POX,

and APX. SOD is localized in almost all cellular compartments and the water–watercycle in chloroplasts. The components of the ascorbate–glutathione cycle arelocalized in chloroplasts, cytosol, mitochondria, and apoplast, while both glutathioneperoxidase (GPX) and CAT are localized in peroxisomes [195].Most studies demonstrated that there is a correlation between the antioxidant

capacity and salt tolerance in different halophytic plant species, includingCentaurea tuzgoluensis [196], P. maritima [197], and Cakila maritima [198]. However,for some species, no significant changes, or even a decrease in activity of someantioxidant enzymes, have been reported (Table 7.2). The effects of salt stress onthe activity of antioxidant enzymes in some halophytes and moderate halophytesare given in Table 7.2. It is well known that halophytes possess higher oxidativestress tolerance than glycophytes. However, as reported by Shabala et al. [199], theseconclusions are derived from comparisons of only a rather limited number ofspecies, such as T. halophila and A. thaliana.

Table 7.2 Effects of salt stress on the activity of antioxidant enzymes in some halophytes and

moderate halophytes.

Species Enzyme Changes Reference

Gypsohila oblan-ceolata

SOD, CAT, APX Increased at 50 and 100mMNaCl

[200]

APX Decreased at 300mM NaClSOD Unaffected at 300mM NaClCAT, POX Increased at 300mM NaCl

Salicornia persica CAT, SOD Increased at 170 and340mM NaCl

[201]

Centaurea tuzgo-luensis

SOD, APX, glutathionereductase

Increased at 300mM NaCl [196]

CAT Unaffected at 300mM NaClHordeum mari-num

SOD, CAT, APX, glutathionereductase

Increased at 300mM NaCl [202]

Salicornia brachi-ate

SOD, POX, APX, glu-tathione reductase

Increased [203]

CAT DecreasedThellungiella halo-phila

SOD Increase [204]

Avicennia marina SOD Increase [205]Plantago mari-tima

SOD, POX Increased at 100 and200mM NaCl

[197]

CAT, APX, glutathionereductase

Increased at 200mM NaCl

APX, CAT Decreased at 100mM NaClglutathione reductase Unaffected at 100mM NaCl

Beta maritima SOD, CAT, POX, APX,glutathione reductase

Increased at 150 and500mM NaCl

[206]

7.3 Effects: Primary and Secondary 177

7.4

Conclusion

In this chapter, we focused on salt stress responses of plants in the light of recentadvances. We tried to evaluate and comment with respect to our currentknowledge about physiological, biochemical, and molecular mechanisms under-pinning the effects of salt stress and tolerance. After giving a definition andgeneral terms, we provided knowledge regarding salt stress perception, signaling,and primary and secondary effects of salinity. Although there exist of several majorcandidates as sensors of salinity, the perception of Naþ ions by plant cells is still amystery. Hence, it seems that this topic is going to be one of the most activeresearch area in the future, as it is present. As for the better-known part of thestory – salt stress signaling – we now know that Ca2þ , pH, ABA, ROS,phosphatidic acid, and IP3 all have important roles in transferring the saltstimulus into the appropriate cellular response via transcriptional induction ofstress-responsive genes or direct regulation of enzymatic activities or channelproteins in the immediate response. Their signaling role in salt stress responsesare all well defined in the literature and, hence, in this chapter. Primer effects ofsalt stress are discussed in two phases: osmotic and ionic – both intensive researchareas. Moreover, we have tried to evaluate salinity-induced oxidative stress asdeeply as possible, even though limited information is available.Salt tolerance research that contributes to our understanding of subjects ranging

from gene regulation and signal transduction to ion transport, osmoregulation, andmineral nutrition also represents an important part of basic plant biology. Itsimportance will inevitably gain ground under global climate change. Hence, plantsalt tolerance studies using information from plant genome sequencing andtransgenics, and integrating them with functional genomics, transcriptomics,proteomics, and metabolomics tools, will undoubtedly continue to enhance ourunderstanding of the mechanisms of salt stress tolerance.

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188 7 Current Concepts about Salinity and Salinity Tolerance in Plants