Reactive oxygen species regulation and antioxidantdefence in halophytes

Rengin OzgurA, Baris UzildayA, Askim Hediye SekmenA and Ismail TurkanA,B

ADepartment of Biology, Faculty of Science, Ege University, Bornova 35100, Izmir, Turkey.BCorresponding author. Email: ismail.turkan@ege.edu.tr

This paper originates from a presentation at the COSTWG2Meeting ‘Putting halophytes to work – genetics, biochemistryand physiology’ Hannover, Germany, 28–31 August 2012.

Abstract. Production of reactive oxygen species (ROS), which are a by-product of normal cell metabolism in livingorganisms, is an inevitable consequence of aerobic life on Earth, and halophytes are no exception to this rule. Theaccumulation of ROS is elevated under different stress conditions, including salinity, due to a serious imbalance betweentheir production and elimination. These ROS are highly toxic and, in the absence of protective mechanisms, can causeoxidative damage to lipids, proteins andDNA, leading to alterations in the redox state and further damage to the cell. Besidesfunctioning as toxic by-products of stress metabolism, ROS are also important signal transduction molecules in controllinggrowth, development and responses to stress. Plants control the concentrations of ROS by an array of enzymatic and non-enzymatic antioxidants. Although a relation between enzymatic and non-enzymatic antioxidant defence mechanismsand tolerance to salt stress has been reported, little information is available on ROS-mediated signalling, perception andspecificity in different halophytic species. Hence, in this review, we describe recent advances in ROS homeostasis andsignalling in response to salt, and discuss current understanding of ROS involvement in stress sensing, stress signallingand regulation of acclimation responses in halophytes. We also highlight the role of genetic, proteomic and metabolicapproaches for the successful study of the complex relationship among antioxidants and their functions in halophytes,which would be critical in increasing salt tolerance in crop plants.

Additional keywords: antioxidant, halophyte, ROS, salinity.

Received 22 December 2012, accepted 3 April 2013, published online 16 May 2013

Introduction

Salinity is a worldwide problem that affects nearly 20% ofagricultural lands (FAO 2000). This common stress reducescrop growth and thereby yields. Plants that can grow andsurvive under high salinity conditions number ~1% ofterrestrial species and these halophytic plants occur as naturalvegetation of saline soils (Rozema and Flowers 2008).Halophytes can complete their life cycle in at least 200mMNaCl (Flowers and Colmer 2008) and tolerate higherconcentrations (e.g. English and Colmer 2013), and manyshow maximum growth in the presence of salt (see Flowerset al. 1977). Because of these attributes, many researchers areinterested in understanding how these plants withstand salt andin being able to transfer this knowledge to crops. Therefore,elucidating adaptive mechanisms (physiology and survivalstrategies) is a first step to achieve ambitious goals of 21stcentury’s agricultural expectations, which are increasing yieldsand survival of crops on saline lands (Vinocur and Altman 2005;Ashraf 2009).

Salinity affects plant growth in several ways, through osmoticand ionic effects (Munns and Tester 2008). Soluble salts in soil

decrease the water potential requiring osmotic adjustment forthe plant to absorb water. As a response to this phenomenon,plants can accumulate ions and other solutes to decrease theirosmotic potential and hence their water potential below thatof the surrounding soil. In this respect, responses by plants todrought and salinity are similar; however, the physical conditionsof the environment are different. Water is limited in soil underdrought, but in saline soils although water is present it is lessavailable for plants to use.

The ionic effects of salinity result from their accumulation inplant tissues, especially leaves. Transpiration is the driving forcethat moves salts from the roots to shoots and as a consequence,this results in accumulation of ions in the leaf cells. The toxiceffects of these ions are seen first in older leaves. As youngerleaves expand, accumulated salts are diluted and this delays theoccurrence of toxic effects of these ions (Munns and Tester2008). However, inhibition of growth due to an osmotic effectremoves this advantage for younger leaves. Halophytic plantsemploy a variety of mechanisms to overcome the toxic effects ofthese ions: (i) entry of Na+ is restricted in the root; (ii) Na+ takeninto cell can be compartmentalised into vacuoles; (iii) Na+ can be

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extruded out of the cells via Na+/H+ antiporter in root epidermalcells and parenchyma cells surrounding the xylem (Blumwaldet al. 2000; Shi et al. 2002; Bartels and Sunkar 2005; Olías et al.2009); or (iv) some halophytes can also use salt glands to balancetheir ion relations (Rozema and Flowers 2008). Hence,halophytes exclude (Hordeum spp., Melilotus spp., Rhizoporaspp.), accumulate (Atriplex spp., Salicornia spp.) or excrete(Distichlis spp., Avicennia spp.) these ions by using one of themechanisms given above or a combination of them to surviveunder saline conditions (Yensen 2008).

In addition to the toxic effects ofNa+ andCl– ions, their excesscan also cause nutritional disturbance in plants by inhibiting theuptake of other ions. Uptake of nutrients such as Ca2+ and K+,were highly reduced by excess Na+ and NO3

+ by Cl– (Hu andSchmidhalter 2005); deficiency of these nutrients caused growthinhibition in glycophytes (Maas and Grieve 1987; Hu andSchmidhalter 2005).

ROS production and oxidative stress in plants

Reactive oxygen species (ROS) are produced under normalconditions as a by-product of metabolism. Although a normaloccurrence, oxidative stress can also occur as a secondarycomponent of other stresses such as salinity. Mechanisms ofROS production and toxicity are common in all plants andhalophytes are not an exception to this. However, theantioxidative capacity of plants is able to balance these ROSand keep them at non-toxic levels. ROS is used as a collective

term for several O2-derived-radicals like superoxide anion(O2*

–), peroxyl radicals (RO2–), alkoxyl radicals (RO*),

hydroxyl radical (HO*) and also for non-radical singlet oxygen(1O2) and O2*

– derived non-radical hydrogen peroxide (H2O2).H2O2 is produced as a result of dismutation of O2*

–, which canoccur spontaneously or enzymatically in the cell or apoplast. Inthe presence of metal ions such as Fe3+, HO* can be produced asa result of Fenton and Haber-Weiss reactions (Kehrer 2000).When a plant faces environmental fluctuations like salinity,metabolic homeostasis is disturbed and production of ROScan drastically increase, causing oxidative stress in the cells.Moreover, the type and amounts of ROS production also varybecause of the wide distribution of metabolic processesthroughout different cell compartments. (Mittler et al. 2004)(Fig. 1).

Under salinity and drought, as a primary response, stomataare closed to reduce loss of water. However, this response alsolimits gas exchange, which is vital for effective photosynthesis.Limitation of gas exchange causes a variety of redox changes inthe cell due to changes in the effectiveness of photosynthesis.Decreased availability of CO2 can be dramatic in terms of ROSproduction when photosynthetic active radiation (PAR) is high.Uncoupling of light reactions and the Calvin-Benson cycle isthe main cause of ROS production in chloroplasts. Whenelectron transport chains are overloaded, 1O2 can be producedin PSII (Asada 2006). The triplet state of PSII, which is producedby excess excitation, transfers its energy to triplet O2 that in turncauses the production of 1O2. A relaxation mechanism to prevent

High concentration of salt in soil

Decrease in water potential

Reduction in stomatal conductance

Decreased CO2 diffusion

Na+CI–

Na+

Na+

Na+

Na+

Na+

CI– CI–

CI–

CO2diffusion

Chloroplasts

ROS Source Scavenger1O2 PSII over excitation α-tocopherol,β-carotene

O2.– PSI Mehler reaction SOD

H2O2 Dismutation of O2.– APX, GPX, Prx

HO. Fenton/Haber-WeissReaction

Flavonoids

Mitochondria

ROS Source Scavenger

O2.– Complex I and III SOD

H2O2 Dismutation of O2.– APX, CAT, GPX, Prx

HO. Fenton/Haber-WeissReaction

Flavonoids

Peroxisomes

ROS Source Scavenger

H2O2 Photorespiration CAT, APX

HO. Fenton/Haber-Weiss Reaction

Flavonoids

Plasmamembrane-apoplast

ROS Source Scavenger

H2O2 Peroxidase,O2

.–dismutationAPX,CAT

O2.– NADPH oxidase SOD

HO. Fenton/Haber-Weiss Reaction

Flavonoids

Uncoupling of light and stromal reactions Increased photorespiration

SignalingΨSoil decrease

dddd

Perturbation of redox balance

Fig. 1. Sites and mechanisms of reactive oxygen species (ROS) production and detoxification in the cell. Abbreviations: APX, ascorbate peroxidase;CAT, catalase; GPX, glutathione peroxidase; Prx, peroxiredoxin; SOD, superoxide dismutase.

B Functional Plant Biology R. Ozgur et al.

production of 1O2 has evolved in plants, which results in theproduction of O2*

– instead of more reactive 1O2. This reaction,which occurs in PSI, is called theMehler reaction (Mehler 1951).Chloroplastic antioxidant enzymes detoxify O2*

– produced bytheMehler reaction to H2O in the ‘water-water cycle’, which willbe explained in detail in this review. The water-water cycle is acritical mechanism to prevent highly reactive 1O2 production thatcan only be scavenged by non-enzymatic antioxidants.

Another ROS-producing process caused by limited gasexchange under salinity is photorespiration. In the first step ofcarbon fixation when ribulose-1, 5-biphosphate carboxylase/oxygenase (Rubisco) uses oxygen as a substrate instead ofCO2, phosphoglycolate is produced. During detoxification andrecycling of phosphoglycolate back to 3-phosphoglycerate,H2O2 is produced in peroxisomes by glycolate oxidase (for adetailed review see Bauwe et al. 2010). Under salt stress, anydecrease in available CO2 increases rates of photorespiration,which increases the production of H2O2 (Corpas et al. 1993;Noctor et al. 2002). Under these circumstances, insufficientantioxidative defence in peroxisomes can increase theoxidative load of the cells causing cellular damage. However,this process is minimised in halophytes using C4 photosynthesisdue to CO2 concentrating mechanism around the Rubisco(Bauwe et al. 2012).

Besides photosynthesis-related ROS, production of ROScan also occur during respiration in mitochondria. Similar tochloroplasts, over reduction of the mitochondrial electrontransport chain can cause production of O2*

–. Electrons canescape from complexes I and III to molecular oxygen if theother electron acceptors in the line are over reduced (Noctoret al. 2007). To prevent over reduction of electron carriers,plants can utilise an alternative terminal oxidase. Thealternative oxidase can take electrons from the ubiquinonepool and transfer them to oxygen to form H2O (Millar et al.2011). This mechanism makes room for electrons moving fromcomplex I and II to the ubiquinone pool and also lowers thereducing pressure on complexes III and IV. In thisway, unwantedROS production in these complexes can be reduced.

There is no evidence for anymechanismof direct ion-toxicity-inducedROS production up to now, other than through effects onphotosynthesis and respiration. However, some hypotheses can

be suggested: (i) excess accumulation of Na+ ions can affect themolecular stability of proteins; (ii) Na+ can compete for K+

binding sites in proteins and affect their activities; (iii) excessNa+ can cause deficiency in uptake of other macro and micronutrients from soil, which are needed for enzyme activity ascofactors; and (iv) Na+ can disturb membrane stability andpermeability causing complications in energetic processes inthe cell. All of these steps given above can disturb cellularhomeostasis and can cause production of ROS in differentcellular compartments.

Among ROS, the most reactive molecules are 1O2 and HO*.These molecules cannot be detoxified enzymatically and theirdetoxification relies on non-enzymatic antioxidants like a-tocopherol and b-carotene. However, as mentioned before,plants use mechanisms to prevent their production like thewater-water cycle (to prevent 1O2) or sequestering of metalions in protein complexes like ferritins (to prevent HO*). IfROS are produced over a certain threshold, they can damagecellular components such as nucleic acids, lipids and proteins.Damage to these molecules can cause severe dysfunctions inthe cell. 1O2 and HO* can cause lipid peroxidation, which startsa chain reaction that can also create reactive products such asaldehydes, ketones, hydroxyl acids (Sunkar et al. 2003). Lipidperoxidation is defined as the oxidative decomposition ofpolyunsaturated lipids in membranes. Peroxidation of lipidscan be induced by three different mechanisms: (i) autoxidation(Halliwell and Gutteridge 1989); (ii) photooxidation (Aro et al.1993); and (iii) enzyme catalyses (lipo- or cyclo-oxygenases)(Feussner and Wasternack 2002). Lipid peroxidation due toROS is a non-enzymatic autoxidation processes. The extent ofperoxidation of membrane lipids has often seen as a means toassess the stress-induced oxidative stress and the degree ofplant sensitivity (Table 1). The level of lipid peroxidationis usually determined by measuring thiobarbituric reactivesubstances (TBARS) or malondialdehyde contents (MDA),which are end- products of lipid peroxidation. In Table 1, saltconcentrations at which increased lipid peroxidation is firstobserved are listed. Excess lipid peroxidation affectsmembrane selectivity and permeability that can cause leakageof ions and other metabolites (Debez et al. 2004; Ben Amor et al.2006). Along with HO*, non-enzymatic lipid peroxidation

Table 1. Halophytes and their resistance to salinity based on occurrence of oxidative stressNaCl concentrations which first significant increase in lipid peroxidation (TBARS content) were observed in the shoots

Species NaCl concentration causingoxidative injury

Duration of treatment References

Sesuvium portulacastrum 1000mM 8 days Lokhande et al. (2011)Atriplex portulacoides over 1000mM 40 days Benzarti et al. (2012)Suaeda salsa 400mM 7 days Qiu-Fang et al. (2005)Salicornia branchiata 400mM 14 d Parida and Jha (2010)Crithmum maritimum over 300mM – Ben Hamed et al. (2007)Hordeum marinum over 300mM 7 days Seckin et al. (2010)Suaeda persica 300mM 45 days Aghaleh et al. (2009)Cakile maritima (Jerba) 200mM 20 days Ben Amor et al. (2006)Spartina alterniflora 200mM – Hessini et al. (2009)Suaeda europaea 200mM 45 days Aghaleh et al. (2009)Gypsophila oblanceolata 150mM 14 days Sekmen et al. (2012)Beta maritima 150mM 6 days Bor et al. (2003)

ROS regulation and antioxidant defence in halophytes Functional Plant Biology C

products can also modify proteins and change their activities ormake them more susceptible to proteolysis.

Aldehydes are one of the major toxic products of lipidperoxidation and removal of these molecules is vital to preventfurther damage. Aldehyde dehydrogenases and aldose/aldehydereductases can catalyse the detoxification of these molecules.Overexpression of these enzymes enhanced salinity tolerance inArabidopsis (Sunkar et al. 2003; Kotchoni et al. 2006). Researchon aldehyde dehydrogenases in halophytes has focussed onglycine betaine aldehyde dehydrogenases and so, productionof the osmoprotectant glycine betaine (Fitzgerald et al. 2009).In halophytes, the role of other aldehyde dehydrogenases andaldose/aldehyde reductases in response to high salinity remainsto be elucidated.

Besides peroxidation of lipid membranes, ROS can alsomodify proteins by oxidation of some amino acid residues.Sulfur containing amino acids, cysteine and methionine, areespecially prone to oxidation. The thiol group of cysteine canbe oxidised to disulfide, sulfenic acid, sulfinic acid and sulfonicacid. Although the first two of these oxidised states are reversibleand widely observed in redox regulation of proteins, sulfinic acidcan be reduced only by sulfiredoxins and oxidation of sulfonicacid is irreversible (Rinalducci et al. 2008). Moreover, Lys, Arg,Pro and Thr can be carbonylated resulting in formation ofreactive aldehyde or ketone groups, and evidence suggests thatthese modifications are irreversible (Shacter 2000). Thesemodifications can cause protein inactivation, crosslinking andeven breakdown (Stadtman and Levine 2000; Gechev et al.2006). In glycophytes, protein oxidation and carbonylation arestudied to detect oxidatively modified proteins using proteomicapproaches and compartments where oxidative damage occurs(Johansson et al. 2004; Job et al. 2005; Qiu et al. 2008). Forexample, Bartoli et al. (2004) found that protein carbonylationis higher in mitochondria as compared with chloroplasts andperoxisomes in wheat under drought. This may be an indicationthat eithermitochondria aremore susceptible to oxidative damageor removal of modified proteins in mitochondria is less efficient.In halophytes, studies investigating oxidative modificationsof proteins are very limited. For instance, although lipidperoxidation levels increased, protein carbonylation did notchange in Hordeum maritimum under salinity (Hafsi et al.2010). However, the salt concentration in this study was ratherlow (100mM) to cause severe oxidative stress for a halophyticspecies. Therefore, in further studies besides peroxidation of lipidmembranes, protein carbonylation is yet to be investigated inhalophytes to gain knowledge of the extent of oxidative proteinmodification in these plants.

From the discussed results, it is evident that lipids and proteinsare the main targets of damage caused by ROS in the cell. In mostof the oxidative-stress-related studies conducted with halophytes,levels of lipid peroxidation were determined by measuringTBARS. However, since reactions with oligosaccharides andpigments such as anthocyanin and other substances includingbetacyanins (Hayakawa and Agarie 2010) can occur in theTBARS assay, an overestimation of lipid peroxidation canoccur. Besides the molecules mentioned above, halophytes canalso accumulate different types of metabolites that mightinterfere with this assay and the extent of their accumulation isnot known for these plants (Lugan et al. 2010). However, the

TBARS assay still remains popular due to its simplicity,cheapness and rapidity (Hodges et al. 1999). In addition toTBARS, electrolyte leakage, direct evidence for membranedamage through increased permeability, can also be used tomeasure the damage caused by ROS to membranes. Usingthese two parameters in conjugation would increase the qualityof data on oxidative injury (Ben Amor et al. 2006; Ben Hamedet al. 2007). Protein oxidation is measured by determination ofcarbonylated proteins,which are irreversibly damaged.However,this assay does not measure the oxidation state of protein boundthiols, which are greatly influenced by the oxidative load ofthe cells. Measurement of oxidation state of protein boundthiols may also be an important parameter to reflect the redoxstate of the cell.

ROS detoxification in halophytes

Although ROS production and toxicity mechanisms are commonin glycophytes and halophytes, detoxification strategies vary inresponse to salinity in regards of total antioxidant activity andtype of isoenzymes expressed.

Enzymatic antioxidants

To both avoid excessive ROS accumulation during stress andmaintain the correct levels of ROS for signalling, plants possess acomplex antioxidant defence system including non-enzymaticantioxidants such as ascorbic acid, glutathione, a-tocopherolsand b-carotenes; and enzymatic antioxidants such as superoxidedismutase (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). The redox regulatory enzymes such as theascorbate-glutathione cycle enzymes monodehydroascorbatereductase (MDAR; EC 1.6.5.4), glutathione reductase (GR;EC 1.8.1.7), glutathione peroxidase (GPX; EC 1.11.1.9),glutathione-S-transferases (GST, EC 2.5.1.18) or the thiolperoxidase type II peroxiredoxin (Prx; EC 1.11.1.15), whichare co-expressed both in the chloroplast and mitochondria(Mittler 2002) are also key components of antioxidant defence.

Several studies showed that halophytes have higherconstitutive antioxidant defence activity as compared withglycophytes. For instance, the halophyte Hordeum marinumhad higher constitutive levels of CAT, POX, APX and GRthan H. vulgare (Seckin et al. 2010). In addition to this, theSOD gene of Thellungiella salsuginea was expressed at muchhigher levels under unstressed conditions than in Arabidopsisunder salinity (Taji et al. 2004). Ellouzi et al. (2011) also showedthat POX, SOD, CAT activities of Cakile maritima were higherthan that of A. thaliana.

Under salinity, new isoenzymes of antioxidant defenceenzymes can be induced and the activities of alreadysynthesised isoenzymes can increase (Table 2). These can be aresponse to production of ROS in different compartments ofthe cell such as chloroplast, peroxisomes or mitochondria.SOD has three different isoenzymes, which are FeSOD, Cu/ZnSOD, MnSOD, which can be distinguished by inhibitortreatments (Vitória et al. 2001). Generally, FeSOD localises inchloroplast, MnSOD in mitochondria and peroxisomes andCu/ZnSOD in chloroplast and cytosol (Alscher et al. 2002).Different regulation of SOD isoenzymes has been observed in

D Functional Plant Biology R. Ozgur et al.

different halophytes (Table 2). For example, in H. marinum,Mn-SOD4 and Cu/ZnSOD7 were only detected under stressconditions (Seckin et al. 2010), whereas in Suaeda salsa nonew SOD isoenzymes were triggered by stress (Qiu-Fang et al.2005).

There are other examples of enzyme induction being usedto maintain a balance between antioxidant enzymes and ROSproduction. For example, activities ofmajor antioxidant enzymessuch as SOD, APX, CAT, GST and GPX can be significantlyincreased in some halophytes. In Sesuvium portulacastrum, 1MNaCl treatment increased SOD, APX, CAT enzyme activities(Lokhande et al. 2011). In contrast, no significant change or adecrease in activity of some antioxidant enzymes were reportedby BenHamed et al. (2007; inCrithmummaritimum). Moreover,in Mesembryanthemum crystallinum, 400mM NaCl treatmentinduced the H2O2 content and but no significant difference wasdetermined in CAT activity, whereas a slight increase inperoxidase activity was observed (Shevyakova et al. 2006).

Strategies of antioxidant defence and response can differbetween halophytes. However, in general these strategies canbe separated in three groups.

(1) Developmental difference (changes occurring duringgermination and the vegetative state), for example, newisoenzymes (one Cu/ZnSOD, three MnSOD) were foundin vegetative plants of Gypsophila oblanceolata whencompared with the isoenzyme pattern during germination(Sekmen et al. 2012). However, in contrast to this, three POXisoenzymes disappeared between germination and thevegetative stage.

(2) Temporal difference, as an example of temporal changes, inCentaurea tuzgoluensis new Cu/ZnSOD isoenzymes (totalof three) were observed as the duration of salinity increased(long-term stress) (Yıldıztugay et al. 2011). In another study,the effect of the duration of NaCl treatment on antioxidantdefence was compared between the halophyte Cakilemaritima and the glycophyte A. thaliana. A significantincrease was found in SOD, CAT, POX activities at 4 hof 400mM NaCl treatment in C. maritima and the highest

levels of mentioned enzymes were observed at 72 htreatment in Arabidopsis under 100mM NaCl (Ellouziet al. 2011). This suggests that antioxidant defenceresponse is more rapid in halophytes as compared withglycophytes. This rapid response to prevent irreversibledamage may be a key trait for tolerance to salt-inducedoxidative stress, before accumulation of ROS above acertain threshold in halophytes. Hence, whether halophytesin general respond faster to salt-induced oxidative stress inregards of antioxidant defence as comparedwith glycophytesis an important topic that needs to be elucidated.

(3) Those depending on type of tissue. for example, differentactivities of antioxidant defence enzymes were detected inroots and shoots of Crithmum maritimum under optimal andhigh salt concentrations: when the highest SOD activity wasobserved in shoots ofC.maritimum, the highestCATactivitywas detected in roots under 50mM NaCl (Ben Amor et al.2005; Ben Hamed et al. 2007).

There are several ways that a plant can avoid production ofROS, for example, via the xanthophyll cycle (to avoid 1O2) inchloroplasts and terminal oxidases in mitochondria andchloroplasts (to avoid O2*

– and therefore H2O2) (Asada 2006;Millar et al. 2011). Each species (or even cultivar) use thesemeasures to a different extent depending on severity ofenvironmental stresses (Loggini et al. 1999; Venema et al.1999; Kong et al. 2001; Misra et al. 2006; Costa et al. 2007).Therefore, the site and amount of ROS formation may differbetween species, causing variability in antioxidant defenceresponses. However, to resist oxidative stress, species have tomaintain an efficient antioxidant capacity. In this respect,components of antioxidant defence can compensate each otherto attain a certain level of scavenging capacity such as differentialinduction of H2O2 scavenging enzymes, APX, POX or CAT. Aclear example of this phenomenon can be observed in a doubleantisense Nicotiana tabacum with supressed expression ofCAT and APX (Rizhsky et al. 2002). We note that this plantwas more resistant to oxidative stress because of the inductionof other compensatory mechanisms such as recycling of Asc

Table 2. Superoxide dismutase (SOD) is an important ROS scavenging enzyme, which is known as ‘first line ofdefence’ against oxidative stress

New isoenzymes in different compartments can be induced by salinity. This table summarises newSOD isoenzymeswhichoccur as a response to salinity in halophytes and major SOD isoenzymes in response to salinity; nd, not determined

Species New isoenzymesunder salinity

Major SODisoenymes

References

Gypsophila oblanceolata MnSOD1,2,3 MnSOD Sekmen et al. (2012)Cu/Zn SOD MnSOD+FeSOD

Centaurea tuzgoluensis Cu/Zn SOD1,4,5 Cu/Zn SOD2,3 Yıldıztugay et al. (2011)Fe SOD 1

Hordeum marinum MnSOD4 MnSOD5 Seckin et al. 2010;Cu/Zn SOD 7 Cu/Zn SOD6

Gypsophila aucheri Mn SOD Mn SOD Sekmen Esen et al. (2012)Suaeda salsa nd Cu/Zn SOD Qiu-Fang et al. (2005)Crithmum maritimum Cu/Zn SOD1 Cu/Zn SOD Ben Amor et al. (2005)Brugeria parviflora nd MnSOD Parida et al. (2004)

CuZnSODFeSOD

Suaeda salsa CuZnSOD CuZnSOD 1,2 Wang et al. (2004)

ROS regulation and antioxidant defence in halophytes Functional Plant Biology E

and chloroplastic alternative oxidase in the absence of theenzymes CAT and APX than the single antisense plants (onlyCAT or APX). However, in halophytes, there is no evidenceof such genomic redundancy and plasticity about antioxidantdefence up to this date.

Non enzymatic antioxidants

Beside the enzymatic antioxidants there are several non-enzymatic antioxidants in plant cells, which are extremelyimportant to prevent the damaging effects of ROS. Ascorbicacid (Asc) and glutathione (GSH) are the most important andcommon non-enzymatic antioxidants and compose the mostparts of non-enzymatic antioxidant pool. Asc and GSH will bediscussed later in this review because of their role in control ofredox state.

The low molecular weight antioxidants also includetocopherols, which scavenge lipid peroxyl radicals and limitthe damaging effects of lipid peroxidation in membranes(Munné-Bosch and Alegre 2002). In glycophytes, there aremany reports of antioxidant activity of tocopherols. In Suaedamaritima seeds, concentration of a-tocopherols was strictlybound with seed viability as protection from oxidative damage(Seal et al. 2010). In addition to this, Ellouzi et al. (2011)compared a- tocopherol levels of Cakile maritima andA. thaliana under salinity. The basal level of a-tocopherol inC. maritima was twice that in A. thaliana. However, tocopherollevels did not change under salinity in C. maritima, althoughthey decreased by 50% in A. thaliana. Yu et al. (2011) alsoreported that a-tocopherol content of Puccinellia tenuiflora wasincreased by salinity as a non-enzymatic antioxidant. Thesereports indicate that ability to regenerate oxidised tocopherol isimportant under salinity.

Just like a-tocopherols, carotenoids are hydrophobicantioxidants that efficiently scavenge 1O2 and peroxyl radicals.Moreover, the carotenoid content of the halophyte Plantagocoronopus was enhanced by increasing salt treatment (Koyro2006). However, the carotenoid content of Salicornia persicaand S. europaea decreased over the range of 100–600mM NaCltreatments that could be related to photoinhibition or ROSformation (Aghaleh et al. 2009). In this study, chlorophyllcontents also decreased, which might suggest suppression ofphotosynthetic activity. All these studies indicate that halophytesmay differ in regards of accumulation of b-carotene dependingon severity of salinity.

Polyphenols are another important group of non-enzymaticantioxidants including flavonoids, anthocyanins and tannins(Smirnoff 2005). They have strong antioxidant capacitydepending on donation of electrons to hydroxyl groups. Thereare many studies on polyphenol contents of halophytes in theliterature (Kalita and Saikia 2004; Meot-Duros andMagne 2009;Trabelsi et al. 2010; Gómez-Caravaca et al. 2012). Ksouri et al.(2009) reported relatively high polyphenol content andantioxidant defence capacity in the medicinal halophyteTamarix gallica. Furthermore, the polyphenol content ofhalophytes can vary in the same genus: Mesembrythemumedule showed greater total phenolic content compared withM. crystallinum and M. nodiflorum (Falleh et al. 2009). Inaddition to this, plants belonging to different ecotypes can also

show differential response in polyphenol accumulation undersalinity. Climatic conditions also affect the polyphenol contentof the same halophyte; although polyphenol accumulation inCakile maritima increased under salinity in plants collectedfrom arid regions, no increase in polyphenols was observedin plants collected from humid regions (Ksouri et al. 2007).Mesembrynthemum edule collected from arid regions had ahigher polyphenol content than plants collected from semiaridregions (Falleh et al. 2012).

Compatible solutes such as proline can be considered asnon-enzymatic antioxidants due to their ability to scavengeHO* (Szabados and Savoure 2010). As a response to salinity,enhanced proline concentrations were reported in halophytessuch as Atriplex halimus (Ben Hassine et al. 2008),Mesembryanthemum crystallinum (Shevyakova et al. 2009)and Suaeda salsa (Wu et al. 2012a). Proline accumulation dueto increasing concentrations of NaCl treatment (up to 500mM)was also enhanced in T. salsuginea and in Arabidopsis thalianaeskimo-1 mutant, which was described as a proline over-accumulating plant that could withstand up to 300mM NaCldue to increased antioxidative capacity (Ghars et al. 2008).However, recently, Signorelli et al. (2013) clearly showed thatproline could not quench 1O2 in aqueous buffer by usingphosphorescence emission of 1O2 and spin trapping EPR.Therefore, these data suggest that the antioxidative attribute ofproline may vary due to type of available ROS, for exampleHO* in that cell compartment.

Although most of studies concentrate on enzymaticscavenging systems, non-enzymatic antioxidants are also vitalbecause they can scavenge ROS that cannot be detoxified byenzymatic systems; ROS like 1O2, HO*. Previously, it has beenshown that these ROS were major causes of lipid peroxidation(Triantaphylides et al. 2008). Once the enzymatic detoxificationsystem is overwhelmed, non-enzymatic antioxidant like a-tocopherols, carotenoids, polyphenols are needed to preventoxidative damage. As mentioned above, these molecules areaccumulated to higher concentrations in unstressed conditionsin halophytes as compared with glycophytes, which iscompatible with higher enzymatic antioxidant activity of theseplants. Therefore, accumulations of these molecules are clearlyadvantageous to prevent damage caused by salinity-inducedoxidative stress.

Redox state and its regulation in halophytes

All plants try to maintain cell homeostasis including their redoxstate as a response to changes in oxidative load. To balance theredox state within certain limits, all plant cells contain redox-active compounds such as ascorbate (Asc), glutathione (GSH)and the pyridine nucleotides, NADH and NADPH as well asproteinaceous thiols (Queval and Noctor 2007). Moreover,recent studies show that induction and/or maintaining thesemechanisms vary between halophytes and glycophytes underdifferent salt concentrations, as indicated below.

Ascorbate/dehydroascorbate (Asc/DHA) pair

Asc and GSH are the most important non-enzymatic ROSscavenging compounds (Colville and Smirnoff 2008). Ascscavenges H2O2 either directly or as substrate of ascorbate

F Functional Plant Biology R. Ozgur et al.

peroxidase (APX), which is one of the enzymes of the ascorbate-glutathione cycle (Asc-GSH cycle). Asc is used as a specificelectron donor by APX to reduce H2O2 to H2O with generationof monodehydroascorbate (MDHA). In this cycle, one Ascand one dehydroascorbate (DHA) can be produced as a resultof dismutation of two MDHA, which can occur spontaneously(Fig. 2), orMDHA can be regenerated back to Asc enzymaticallyby NAD(P)H-dependent monodehydroascorbate reductase(MDHAR). Moreover, DHA can also be converted to Asc bydehydroascorbate reductase (DHAR) that uses GSH as anelectron donor.

It is known that Asc together with DHA (Asc : DHA ratio)are important indicators of the redox status in the cell. BesidestheROS-scavenging role ofAsc, the role ofAsc/DHApairwithinthe cellular stress response network is complex (Potters et al.2010). In the halophyte Cakile maritima (cv. Jerba), under saltstress, whereas Asc concentration was gradually decreasedthrough increased APX activity, the Asc : DHA ratio increasedat 100mM NaCl, but decreased at the highest NaCl level(400mM NaCl) (Ben Amor et al. 2006). In contrast, Cai-Honget al. (2005) showed that in the halophyte Suaeda salsa powerfulremoval of H2O2 was possibly performed by a co-operativeupregulation of the H2O2-scavenging system including Ascand GSH contents, GR and APX activities under high salinity.

Glutathione/glutathione disulfide pair (GSH/GSSG)

In the Asc-GSH cycle, GSH is regenerated by GR (NADPHdependent oxido-reductase) as a result of reduction ofglutathione-disulfide (GSSG) produced by DHAR. GSH,which is termed as ‘ubiquitous’ or ‘universal redox buffer’,plays an important role in several physiological processesincluding regulation of sulfate transport, signal transduction,several growth and development related events in plantsincluding cell cycle, cell death and senescence (Noctor et al.1998; Aslund and Beckwith 1999; Ogawa 2005; Kranner et al.2006).GSH is also a potential scavenger of 1O2 andH2O2 (Noctorand Foyer 1998).

GSH takes part in removal of not only excess H2O2 but alsolipid peroxides and detoxification of heavy metals andxenobiotics (Rausch et al. 2007). In removal of H2O2, GSH isused in the Asc-GSH cycle. However, removal of lipid peroxidesis catalysed by GSH S-transferases (GST). GSH together withGSSG (GSH/GSSG pair) are responsible for maintaining thenormal reduced state of cells under stress due to their involvementin redox signalling (Schafer andBuettner 2001; Foyer andNoctor2005). In this signal network, GSH interacts with ROS, redoxmolecules such as thioredoxins (Trxs), glutaredoxins (Grxs), andplant hormones such as salicylic acid (SA) and abscisic acid(ABA).

The glutathione redox state is an indicator of oxidative stresscaused by environmental factors, therefore, it indirectly reflectsthe severity of stress (Potters et al. 2010). For example, inmangrove (Bruguiera parviflora), high salinity (100–400mM)did not changeGSH level, but it significantly decreased (~3-fold)GSSG level. Therefore, the rate of GSH :GSSG was increasedby both increasing concentration ofNaCl, and duration of salinity(Parida et al. 2004). However, in Cakile maritima (cv. Jerba), acoastal halophyte,GSH level, was increasedwhereasGSSG levelremained unaffected (Ben Amor et al. 2005). GSH :GSSG ratiofollowed the same pattern as total GSH concentration. On theother hand, in Hordeum maritimum, an annual halophytic grass,salt stress and K+ deficiency did not change the GSH and GSSGconcentration and GSH :GSSG ratio, indicating that glutathionemetabolism was unaffected, although Asc-GSH cycle wasfunctioning (Hafsi et al. 2010). Moreover, Alhdad et al.(2013) showed that the total glutathione (GSSG+GSH) andGSH levels of Suaeda maritima increased by saline flooding.Therefore, it seems that maintenance of GSH :GSSG ratio isan important trait under oxidative stress caused by salinity. Asmentioned above, plants can either efficiently regenerate theoxidised glutathione (by using GR) or regulate synthesis ofnew GSH to maintain glutathione redox state.

NAD(P)+ and NAD(P)H

NAD and NADP are particularly important in reactions relatedto lipid and nucleic acid synthesis, nitrate reduction, pollendevelopment, pollen maturation and pollen tube growth(Hashida et al. 2009) as well as stress-related signaltransduction (Noctor et al. 2006). They act as a cofactor inthese processes; they do not function as redox buffers (Noctor2006). NADPH and NADH act as reductants for MDHARthat uses electrons originating from NADH. GalDH (L-galactose dehydrogenase), an enzyme involved in synthesising

NADP+

2 GSH GSSG

Asc

AscH2O2

2 H2O

DHA

MDHA NADPH

NADP+

Asc

NADPH

GR

DHAR

MDHARAPX

Fig. 2. Ascorbate (Asc)-glutathione (GSH) cycle. Abbreviations: H2O2,hydrogen peroxide; Asc, ascorbate; APX, ascorbate peroxidase; DHA,dehydroascorbate; DHAR, dehydroascorbate reductase; GSSG, reducedglutathione; GR, glutathione reductase; MDHA, monodehydroascorbate;MDHAR, monodehydroascorbate reductase; NADPH, nicotineamideadenine dinucleotide phosphate.

ROS regulation and antioxidant defence in halophytes Functional Plant Biology G

Asc also is specific for NAD+ (Gatzek et al. 2002). The activity ofGalLDH (L-galactono-1,4-lactone dehydrogenase) is stimulatedby increases inNAD(P)+/NAD(P)H (Wolucka andVanMontagu2003), which suggest a feedback to Asc synthesis underconditions where NAD(P)H is depleted (i.e. by increased GRactivity). In contrast, NADH status is an important factor inmitochondrial ROS production and cytosolic NADPHprovides the reductant for production of O2*

– (and H2O2 dueto O2* dismutation) by NAD(P)H oxidases. NADK1 (NADkinase-1) and NADK3, which are NADP synthetic enzymes,associated with stress tolerance, are induced by environmentalstresses. NAD(P)+-dependent oxidation and NADPH-dependentreduction catalysed by aldehyde dehydrogenase are importantdetoxification of oxo-derivates of lipid peroxides. Byconsidering the importance of these molecules for maintainingcellular homeostasis during stress it is important to elucidatehow halophytes regulate their synthesis and their reduction state.

Thioredoxins (Trxs)

Trxs are ubiquitous proteins. There have conserved cysteineresidues with the general motifs –C–G–P–C– in their activesites and are reduced by different electron donors. AlthoughTrxs in chloroplast are reduced by a ferredoxin-dependentthioredoxin reductase (FTR), Trxs in the cytosol andmitochondria are reduced by an NADPH thioredoxin reductase(NTR) (Vieira Dos Santos andRey 2006). Trxs play an importantrole in plant tolerance to oxidative stress. They prevent proteinoxidation and lipid peroxidation under stress as a supplier ofreducing power to reductases. Moreover, Trxs act also ascomponents of signalling pathways in the plant antioxidantnetwork or as modulator of ROS scavenging enzymes (Meyeret al. 2008). For now, identification and functional charactersof halophytic Trxs are not clear, but many thioredoxin-likehomologue genes can be found from expressed sequence tag(EST) databases of halophytes (Baisakh et al. 2008; Chen et al.2012) and further investigations are needed.

Peroxiredoxins (Prxs)

Prxs, one of the groups of thioredoxin-dependent reductases,are crucial components of ROS signalling. They act to mediateH2O2homeostasis, to scavenge hydroperoxides, and are involvedin redox reactions. A unique Prx Q, gene was identified underseveral stress conditions in S. salsa and was suggested to beinvolved in H2O2 dependent signalling cascades in chloroplasts(Guo et al. 2004). Also in the halophyte Arthrocnemummacrostachyum, a Prx has been identified recently (Trottaet al. 2012), and the amount of Prx increased when saltconcentration was higher or lower than optimal. It is alsoknown that Prxs are the only enzymes that can scavengeperoxynitrites (ONOO–), which can be produced by reactionof O2

*– with nitric oxide (NO) (Valderrama et al. 2007).

Glutaredoxins (Grxs)

There are conserved cysteine residues with the glutathione-reducible motifs –C–x–x–C in the active site of dithiol-Grx or–C–x–x–S– in the active site of monothiol-Grx (Rouhier et al.2004; Gelhaye et al. 2005). Grx use GSH as an electron donorto catalyse disulfide reactions in the presence of NADPH and

GR, which links its regulation to GSH pool i.e. cellular redoxstate (Meyer and Hell 2005). Grxs play key roles in maintainingthe cellular redox balance. Moreover, Grxs play a role in redox-dependent signalling by reversible protein S-glutathionylation.Glutathionylation is a post-translational modification processthat, briefly, constitutes a disulfide bond between proteins thiolresidues and GSH. Under oxidative stress, increasing productionof ROS brings the protein modification due to changes in thiolresidues of proteins, which is highly damaging in a cell.Glutathionylation prevents the cell from suffering irreversibleprotein damage. Cu/ZnSOD, can catalyse the glutathionylationof Trx and Grx to regulate their activity (Michelet et al. 2005;Dalle-Donne et al. 2007). Glutathionylation targets in halophytesstill need to be elucidated; although ESTs (for T. salsuginea,Zhang et al. 2008) of these genes have been identified, there isno study investigating the regulation of Grxs in detail undersalinity. In addition to this, studies covering cellular GSH redoxstate and responses of Grx are needed to further understandtheir role in salinity tolerance.

ROS signalling in halophytes

Besides the damaging effects caused by oxidation of vitalmolecules like proteins, nucleic acids and lipids, ROS are alsoimportant signal molecules that can act to trigger gene expressionand antioxidant defence machinery. The full picture of ROSsignalling is complex and it is still not well understood. It isrelated to Ca2+ and Ca2+ binding proteins like calmodulin, GTP-binding (G) proteins, phospholipid signalling and activation ofMAPKs (Mittler et al. 2004). A well-known and characterisedsignalling role of ROS is its relationship with abscisic acid(ABA) and stomatal closure. Perception of ABA in guard cellsinducesH2O2 production viaNADPHoxidase that activates Ca2+

permeable channels in the plasma membrane (Kwak et al. 2003).As a result of Ca2+ influx, other processes resulting in stomatalclosure are triggered. Ca2+ homeostasis is a crucial factor thataffects the adaptationmechanism in plants and it is closely relatedto ROS. A detailed review of the interaction between Ca2+ effluxsystems and stress signalling has been published (see Boseet al. 2011).

Recently, a signalling node that includes SOS2 (a kinasephosphorylating SOS1) and H2O2 interacting with nucleosidetriphosphate kinase 2 (NDPK2) has been suggested (Verslueset al. 2007). This relationship is exciting because it can providea connection between the salt overly sensitive (SOS) pathwayand ROS signalling. In this study (Verslues et al. 2007), it wasshown that SOS2 can interact with NDPK2, a kinase that canstimulate MAPK3 and MAPK6, which are key components ofphosphatidic acid signalling inducing responses to oxidativestress (Mittler et al. 2004; Verslues et al. 2007). In addition, itwas also shown that SOS2 can interact with CAT2 and CAT3.These results suggest that response to salt stress is directlyrelated to H2O2-dependent signalling. Moreover, Ohta et al.(2003) provided evidence for SOS2 and ABA insensitive 2(ABI2) interaction, which also suggests an interaction betweenABA signalling and SOS pathway. Previously, it was shownthat GPX and ABI2 can form a protein complex to ensureefficient ABI2 activity by scavenging H2O2, which can inhibitthe activity ofABI2. Interaction betweenSOS2 andABI2 implies

H Functional Plant Biology R. Ozgur et al.

a similar protein complex that may include SOS2, ABI2 andCAT. These studies above were performed in Arabidopsis.However, studies on the relationship between the SOSpathway, especially SOS2 and ROS signalling, in halophytesare limited. In Hordeum brevusubulatum, overexpression ofHbCIPK2, a putative homologue of SOS2, in transgenicA. thaliana deficient in SOS2–1, conferred salt tolerance (Liet al. 2012). From the EST database of Suaeda salsa, ahomologue of Arabidopsis MAPK3 gene, and a putativecalmodulin-binding protein, which was known as a part ofROS signalling was recorded (Zhang et al. 2001). Theseinteractions mentioned above are summarised in Fig. 3.Through the increase in availability of genetic tools and datafor halophytes, it should be possible to investigate thesemechanisms in detail for these salt-adapted species. In

addition, yeast two hybrid interaction screens combinedwith biochemical studies such as conducted by Verslues et al.(2007) can elucidate the protein interactions in the SOSpathway and their possible connections to other pathways inhalophytes.

Among ROS signalling components, G proteins induce ROSproduction in plant cells and are considered the first componentof stress induced oxidative burst in Arabidopsis (Joo et al.2005). Salinity-induced GTP binding proteins can be found inEST databases of many halophytes such as Suaeda salsa,S. maritima, Spartina alterniflora, Thellungiella salsuginea(Zhang et al. 2001; Gong et al. 2005; Baisakh et al. 2008;Sahu and Shaw 2009), but further investigations are neededto understand the interaction between G proteins and ROSsignalling in halophytes.

Possible role of CAT in a protein complexincluding ABI2, SOS2, and CAT;CAT can protect ABI2 from H2O2 inactivation.

Connection with ABA signalling(Ohta et al. 2003)

Connection with ROS signalling

PPI motif

Kinase domain FISL motif

Regulatory domain

SOS3

NDPK2

MAPK3

MAPK6

shoots H2O2

(Moon et al. 2003)

(Halfter et al. 2000)

(Verslues et al. 2007)

CAT 2

CAT 3

SOS2

ABI2

Salt stress

Cytoplasmic calciumsignal elicited by salt stress

Ca+2

SOS1

RCD1Regulation of oxidativestress responses

roots interaction between C-terminaltail of SOS1 and RCD1

(Katiyar-Agarwalet al. 2006)

(Verslues et al. 2007)

unkn

own

inte

ract

ion

site

(Qiu et al. 2002)

Regulation of oxidative

stress responses

Fig. 3. Putativemodel for salt overly sensitive (SOS) pathway components and reactive oxygen species (ROS) interaction. Protein interaction studies suggest asignalling hub focussed around SOS2 protein, which is expressed in both roots and tissues (Liu et al. 2000). Thewell-knownSOSpathway includes interaction ofCa+2 activatedSOS3withSOS2onFISLdomain andactivationof SOS1Na+/H+ antiporter by this complex (Halfter et al. 2000;Qiu et al. 2002). SOS2can interactwith NDPK2 on the same FISL domain (Verslues et al. 2007). NDPK2 is a regulator of MAPK3 and 6 which are known to regulate oxidative stress responses(Moon et al. 2003). SOS2 can also interactwithCAT2andCAT3 in a stress dependentway (Verslues et al. 2007). In the absence of stress this interaction cannot beobserved. Interaction with SOS2 and CAT suggests a role in ROS signalling. SOS2 and ABI2 interaction (on PPI motif) also implies a connection between SOSpathway andABA signalling (Ohta et al. 2003). In this interaction CATmay play a role to create a H2O2 depleted zone aroundABI2 to regulate its activity (H2O2

inhibits ABI2). C-terminal tail of SOS1 can also interact with RCD1 which is also a regulator of oxidative stress responses (Katiyar-Agarwal et al. 2006).Abbreviations: ABI, aba insensitive; CAT, catalase;MAPK,mitogen activated protein kinase; NDPK, nucleoside diphosphate kinase; RCD, radical induced celldeath; SOS, salt overly sensitive.

ROS regulation and antioxidant defence in halophytes Functional Plant Biology I

Other roles of ROS

In addition to signal transduction, ROS has also been shown tobe important for transport of Na+ ion in Arabidopsis undersalinity. Inhibition of the ROS-producing enzyme NADPHoxidase by diphenyleneiodonium (DPI) caused decreasedstability of SOS1 mRNA (Chung et al. 2008). SOS1 is aplasma membrane Na+/H+ antiporter that is preferentiallyexpressed in epidermal cells at the root tip and in parenchymacells at the xylem/symplast boundary of roots, stems, and leavesin Arabidopsis, which plays an important role in removal ofNa+ ions from the cell (Shi et al. 2000; Shabala and Mackay2011). Overexpression of SOS1 increased salt tolerance ofArabidopsis (Shi et al. 2002). In the halophyte T. salsuginea(halophila), disturbance of SOS1 caused this plant to turn intoa plant as sensitive to Na+ as Arabidopsis, which showed thevital role of this antiporter in salt tolerance (Oh et al. 2009).A comparative study between two Thellungiella species(T. salsuginea and T. parvula) and Arabidopsis showed thatalthough coding regions are conserved there are distinctivedifferences in 50 untranslated proximal regions of SOS1 (Ohet al. 2010). The authors hypothesised that this suggests ahalophytic transcript structure for SOS1. However, it is notknown how this difference affects mRNA stability inThellungiella or whether this plant also needs ROS to stabiliseSOS1 mRNA as does Arabidopsis. If ROS were also neededfor stabilisation of SOS1 mRNA, the site of production andmode of action of this ROS needs to be elucidated to ensurecomplete understanding of regulation of SOS1 expression inT. salsuginea.

Another role other than ion transport of SOS1 is its interactionwith RCD1 (radical induced cell death 1), which is a regulatorof oxidative stress (Belles-Boix et al. 2000; Fujibe et al. 2004;Jaspers and Kangasjarvi 2010). RCD1 can localise in nuclei andcytoplasm. In a nucleus, RCD1 stimulates some common stress-related transcription factors under oxidative stress. However,under oxidative stress, RCD1 moves to the cytoplasm(Katiyar-Agarwal et al. 2006). Katiyar-Agarwal et al. (2006)showed that the C-terminal tail of SOS1, located in plasmamembranes, can interact with RCD1. This is more directevidence for interaction between ROS signalling and SOSpathway (Fig. 3).

As it can be seen from the examples given above, majorstudies related to ROS signalling are based on glycophytes.Extended studies are needed on the SOS pathway and otherhalophytic adaptive mechanisms to elucidate the roles of ROSsignalling in a halophytic style of life. In this respect, use ofmodel halophytes like Thellungiella should accelerate thisprocess.

Proteomic and transcriptomic approaches to reactiveoxygen species regulation studies on halophytes

Detailed proteomic studies are needed to understand tolerancemechanisms of plants related to ROS regulation, but these arerather limited for halophytes (Kosová et al. 2011) (Table 3.). Thismay be a result of difficulties in growing some halophytes inlaboratory conditions and interference of substances accumulatedby some halophytes with protein extraction and separation. In aChinese halophytic shrub (Nitraria sphaerocarpa), widescreening of protein profiles were performed and 19% of totalidentified proteins were found to involved in antioxidativemechanisms such as glutathione-S-transferases (GST),thioredoxins (Trxs) and oxidoreductases (Chen et al. 2012). Inaddition to this, different protein profiles were determined undervaried salt concentration in halophyte Salicornia europaea. Inthis study, 10 of 111 identified proteins were directly related todetoxification and antioxidant system such as POX, SOD andtheir expression was increased by NaCl accumulation (Wanget al. 2009). In another halophyte, Suaeda aegyptiaca, salinityinduced or suppressed protein expression and seven of 27identified proteins were representative of oxidative stress(Askari et al. 2006).

Other proteomic studies have compared halophytes withclosely related glycophytes. In this context, Pang et al. (2010)compared A. thaliana and its relative T. salsuginea under salineconditions. Five of 32 identified proteins of Thellungiella werepart of antioxidant defence system, whereas only one of 79identified proteins of Arabidopsis was related to antioxidantdefence. Although proteomic studies in halophytes generallyuse total protein extracts of tissues, use of organellarproteomics can further increase our knowledge in halophyticsalt tolerance.

To determine the differences in gene expression betweenglycophytes and halophytes EST databases are particularlyuseful. EST databases of halophytes have been constructed,which include those of Aeluropus littoralis, Suaeda salsa,Spartina alterniflora, Salicornia brachiata, Mesembryanthemumcrystallinum, Halostachys caspica, Avicennia marina (Zhanget al. 2001; Kore-eda et al. 2004; Mehta et al. 2005; Zouariet al. 2007; Baisakh et al. 2008; Jha et al. 2009; Liu et al.2012). From such data it has been shown that antioxidantrelated GST of A. littoralis is upregulated with 300mM saltstress Ts (Zouari et al. 2007). In another halophyte, Halostachyscaspica, GPX and GST genes were upregulated by salt stress(Liu et al. 2012). Furthermore, from EST database of Suaedasalsa, ROS scavenger related genes (catalase, glutathioneperoxidase, ascorbate peroxidase) were identified (Zhang et al.2001).

Table 3. Proteomic studies which identified antioxidant related proteins in halophytes

Species Proteomic studies References

Nitraria sphaerocarpa Total of 19% of identified proteins related to oxidative stress includingglutathione-S transferases, thioredoxins, oxidoreductases

Chen et al. (2012)

Salicornia europaea Ten of 111 identified proteins related to antioxidative mechanisms includingperoxidase, superoxide dismutase

Wang et al. (2009)

Suaeda aegyptiaca Seven of 27 identified proteins were oxidative stress related Askari et al. (2006)Thellungiella salsuginea Five of 32 proteins identified were related to antioxidant defence system Pang et al. (2010)

J Functional Plant Biology R. Ozgur et al.

In the early 2000s, T. salsuginea was proposed to thescientific community as a model halophytic species because ofits 90% gene sequence similarity to Arabidopsis thaliana, itsshort life cycle, self-pollination and salinity tolerance up to500mM NaCl (Bressan et al. 2001; Zhu 2001). Wang et al.(2004) published an EST database of T. salsuginea and identified1178 ESTs; 14% of these EST’s were related to stress defenceincluding ROS scavengers like POX, APX, CAT, GPX, GR andGST. From the results, CAT3 was indicated as one of the mostabundant mRNAs, which indicates the crucial role of thisenzyme for normal growth. In 2008, Zhang et al. published acomparison of transcripts of Thellungiella under saline and non-saline conditions using time-course analysis and found nineputative antioxidant enzymes including POX, APX, DHAR,GR, CAT, and Trx.

A comparison of microarrays between T. salsuginea andA. thaliana under salinity stress has been published by Gonget al. (2005). From this wide study, it is clear that many redoxhomeostasis genes such as GPX, GST and several genes of Gproteins were upregulated in both species, whereas some ofredox homeostasis genes (AtTRX4, ATGSTU20) were onlyinduced in Thellungiella. There was also a significant increaseof transcript intensity in a GPX (ATGPX4) and a GST (AtGSTU26) in T. salsuginea.

Conclusion

In halophytes, studies on ROS to date have been focussed mainlyon detoxification mechanisms (SOD, CAT, POX, APX, GR,ascorbate, glutathione), which are related to salt tolerance.However, studies on regulatory and signalling roles of ROSare rather limited. Hence, most of the data concerning ROSregulation and signalling are mainly based on findingsobtained from glycophytes such as Arabidopsis. Moreover,signalling components (G proteins, MAPK) or and functionsof redox-active compounds (thioredoxin, glutaredoxin,peroxiredoxin) in halophytes are still not well understood.Therefore, extended studies are needed on these subjects usinghalophyte models such as Thellungiella. Relevant data obtainedfrom Arabidopsis, will be helpful to enlighten similarmechanisms for salinity tolerance in Thellungiella.

The complete genome of extremophile T. parvula, which canstand higher NaCl concentrations than T. salsuginea has beenpublished and this genome gives great opportunity to comparegenomic differences between a glycophyte, Arabidopsis, and aclosely related halophyte, and to understand defencemechanismsagainst salt stress (Dassanayake et al. 2011). Since then, thewholegenome of T. salsuginea has also been published (Wu et al.2012b). Hence, completed genome data of T. parvula alongwith T. salsuginea are now valuable tools to evaluate andimprove our understanding of salt stress tolerance. In additionto these developments, by using pyro sequencing, it would beeasier to characterise transcriptomics of halophytes and so toelucidate the full picture of halophytic tolerance mechanisms.

The involvement of ROS in salt tolerance mechanismsof monocotyledonous halophytes is still a mystery. AlthoughThellungiella seems to be an important dicotyledonoushalophytic source for studies on ROS regulation andsignalling, a monocotyledonous halophytic model is also

needed for elucidation of differences between dicots andmonocots (Byrt and Munns 2008). Data that will be obtainedon such a model may also be helpful for transferring thisknowledge to crop plants, the most important of which are allmonocots.

As reviewed here, there is a reasonable amount of data onsome components of antioxidants (activities of antioxidantenzymes and contents of non-enzymatic antioxidant such asascorbate and glutathione) in the current literature. However,especially in halophytes, research on other components ofantioxidant systems (enzymes like GRX, PRX; TRX, low-molecular weight antioxidant molecules, oxidation status ofcysteine) and links between antioxidants and ROS signallingare few. In their pioneering review, Loiacono and De Tullio(2012) suggested the use of a subset of omic disciplines(metabolomics, transcriptomic, proteomic and phenomics) tostudy ROS/antioxidant interaction (antioxidomics), whichwould provide a comprehensive picture of ROS/antioxidantand stress tolerance. The same approach could be used toclarify the role of ROS and antioxidant relationship inhalophytic stress tolerance.

Acknowledgements

We would like to thank Professor Timothy J Flowers for his critical reviewof the manuscript.

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