Superoxide dismutase isozyme activity and antioxidant responses of hydroponically cultured Lepidium sativum L. to NaCl stress

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1 Journl of Plnt Intertions ISSN: (Print) (Online) Journl homepge: Superoxide dismutse isozyme tivity nd ntioxidnt responses of hydroponilly ultured Lepidium stivum L. to NCl stress Arfet Mn, Hjer Mimouni, Amel Terrs, Frh Cheil, Slm Wsti, Emn Ghri & Hel Ben Ahmed To ite this rtile: Arfet Mn, Hjer Mimouni, Amel Terrs, Frh Cheil, Slm Wsti, Emn Ghri & Hel Ben Ahmed (214) Superoxide dismutse isozyme tivity nd ntioxidnt responses of hydroponilly ultured Lepidiumstivum L. to NCl stress, Journl of Plnt Intertions, 9:1, , DOI: 1.18/ To link to this rtile: Tylor & Frnis Aepted uthor version posted online: 4 Ot 213. Pulished online: 25 Ot 213. Sumit your rtile to this journl Artile views: 1368 View Crossmrk dt Citing rtiles: 4 View iting rtiles Full Terms & Conditions of ess nd use n e found t

2 Journl of Plnt Intertions, 214 Vol. 9, No. 1, , RESEARCH ARTICLE Superoxide dismutse isozyme tivity nd ntioxidnt responses of hydroponilly ultured Lepidium stivum L. to NCl stress Arfet Mn *, Hjer Mimouni, Amel Terrs, Frh Cheil, Slm Wsti, Emn Ghri nd Hel Ben Ahmed Unité d Eologie Végétle, Déprtement de Biologie, Fulté des Sienes de Tunis, Université Tunis El Mnr, 292, Tunisi; Groupe de Reherhe en Physiologie Végétle (GRPV), Erth nd Life Institute Agronomy (ELI-A), Université tholique de Louvin, 5 (Bte L ) Ple Croix du Sud, 1348 Louvin-l-Neuve, Belgium (Reeived 13 August 213; epted 28 Septemer 213) The present study ws foused to ssess the physiologil ehvior nd ntioxidnt responses of the mediinl plnt Lepidium stivum L. (ommonly lled Grden ress) sujeted hydroponilly to NCl stress during its vegettive growth stge. The results showed tht the ddition of NCl to growth medium signifintly redued plnt growth. The mgnitude of the response ws lso linked to the plnt orgn onsidered nd NCl onentrtion supplemented to the medium. Tissue hydrtion seemed unffeted y slinity. Redution in dry weight (DW) prodution ws ssoited with high umultion of N + nd Cl nd signifint redution of K + ontent in shoots. The umultion of osmoregultory ompounds (proline nd totl sugrs) in shoots nd roots ws gretly inresed y NCl. Ativity stining of ntioxidnts fter ntive polyrylmide gel eletrophores (PAGE) showed four superoxide dismutse (SOD) isozymes in the extrt of lef-solule proteins (one Mn-SOD, two Fe-SODs, nd one CuZn-SOD), nd three isoforms in roots (Mn-SOD, Fe-SOD, nd CuZn-SOD). Four peroxidse (POD) isozymes in the roots nd only one isozyme in the leves were deteted. The work demonstrted tht tivities of ntioxidnt defense enzymes hnged in prllel with the inresed slinity. In summry, these findings proved tht L. stivum n e lssified s modertely tolernt plnt to slinity. Keywords: Lepidium stivum; NCl; physiologil ehvior; osmoregultory ompound; ntioxidnt enzymes Introdution The mediinl plnts re of gret interest due to their importne in the humn helth. Lepidium stivum L. ommonly lled Grden ress is polymorphi speies. Grden ress is n nnul her, elonging to Brssiee fmily (Kirthikr 1952). L. stivum is polymorphous speies nd elieved to hve originted primrily in the highlnd regions of Ethiopi nd Eritre. It is known for its mediinl vlue s soure of mediinl ompounds. The leves of Lepidium re ntisoruti, diureti, nd stimulnt. The seeds re perients, diureti, toni, demulent, phrodisi, ruefient, rmintive, gltgogue, nd emmengogue (Ndkrni 1954; Diwkr et l. 21; Rehmn et l. 212). It is supplemented in the diet of ltting women to inrese the milk seretion during postntl period (Dtt et l. 211) nd reommended for dirrhe nd dysentery. The roots re itter, rid, nd useful in the tretment of seondry syphillis nd tenesmus nd used s ondiment (Uphof 1959). The queous extrt of L. stivum L. seeds exhiits hypoglyemi tivity oth in norml nd dieti rts without ffeting insulin seretion (Eddouks et l. 25). In mny ountries, this speies is grown in rid nd semirid regions where high onentrtion of slts, minly hloride sodium (NCl), is n importnt hrteristi of the soils whih hs mjor impt on plnt prodution nd produtivity (Zhng & Blumwld 21; Sirm et l. 22; Zhu 22; Jun et l. 25). Plnts re stressed in three wys in sline soils: (1) low wter potentil of the root medium leds to wter defiit, (2) the toxi effets of the ions minly N + nd Cl, (3) nutrient imlne y depression in uptke nd/or shoot trnsport. The result is wide vriety of physiologil nd iohemil hnges in plnts tht inhiit growth nd development, redue photosynthesis, respirtion nd protein synthesis, nd disrupt nulei id metolism (Levine et l. 199; Zhng & Blumwld 21; Sirm et l. 22). One of the iohemil hnges ourring when plnts re sujeted to slt stress is the umultion of retive oxygen speies (ROS) suh s superoxide (O 2 ), hydrogen peroxide (H 2 O 2 ), nd hydroxyl rdils (OH ) (Vn Breusegem et l. 21). ROS n seriously disrupt norml metolism through oxidtive dmge to lipids, protein, nd nulei ids (Meloni et l. 23) nd dmge memrne funtion (Gómez et l. 24). Plnts n regulte the ROS y svenging them with ntioxidnt enzymes suh s superoxide dismutse (SOD), tlse (CAT), nd peroxidses (Bno et l. 213). The existene of multiple moleulr forms of ntioxidnt enzymes nd ny hnges they my undergo in response to vrious environmentl signls imply potentil roles for these isozymes in the detoxifition of ROS (Pinhero et l. 1997). The isozymes ould e used s iohemil mrker to study the tolerne of plnt to stress (Zhng et l. 213). *Corresponding uthor. Emil: mnrfet@gmil.om 213 Tylor & Frnis

3 Journl of Plnt Intertions 441 The eluidtion of physiologil nd iohemil mehnisms is ritil efore trying to introdue geneti nd environmentl improvements to slt stress (Ashrf & Hrris 24; Mn et l. 213). Slt-tolerne mehnisms re quite omplex, inluding osmoti djustment, omprtimenttion of toxi ions (Munns & Tester 28), metolite umultion, ion homeostsis, redox ontrol, nd svenging of tivted oxygen speies (Gill & Tutej 21). Severl studies look t plnt response to slt stress ftors nd ontriute to etter understnding of physiologil mehnisms underlying plnt stress response. These reserhes in reltion to the effet of slinity hve mostly een rried out on griulturl, forge, nd fuel wood speies. However, it ppers tht little informtion is ville regrding the effet of slinity on the growth nd produtivity of mediinl plnts. L. stivum L., Linum usittissimum L., Plntgo ovt Forssk, nd Trigonell foenum-greum L. hve een evluted nd proved to e modertely slt tolernt t germintion nd seedling growth stge (Muhmmd & Hussin 21). The im of this study ws to investigte, for the first time, the physiologil nd ntioxidnt responses of L. stivum, mediinl plnt sujeted hydroponilly to NCl stress t their vegettive growth. Mterils nd methods Plnt mteril nd growth onditions Seeds of L. stivum were germinted in ommeril pet irrigted with fully nutrient solution nd were inuted under fluoresent light (9 µmol m 2 s 1 with 16 h photoperiod t 25 C). When germinted, seedlings were trnsferred to growth hmer (25 C/7% reltive humidity during the dy nd 2 C/9% reltive humidity during the night; photoperiod: 16 h dily with light irrdine of 15 µmol m 2 s 1 ). They were grown in dpted Hoglnd solution (Hoglnd & Arnon 195), ontinuously erted ontining: KNO 3 3 mm, C(NO 3 ) 2 1 mm, KH 2 PO 4 2 mm, MgSO 4.5 mm, Fe-Ethylenediminetetreti id (EDTA) 32.9 µm, nd mironutrients: H 3 BO 4 3 µm, MnSO 4 5 µm, CuSO 4 1 µm, ZnSO 4 1 µm, nd (NH 4 ) 6 Mo 7 O 1 µm for 1 dys. At this time, five onentrtions of NCl were pplied: (ontrol), 5,, 15, nd 2 mm. To void osmoti shok, NCl onentrtions were inresed grdully y 25 mm every dy until the desired onentrtion ws rehed. The nutrient solutions were repled eh three dys. The ph of the nutrient solution ws djusted eh time to 5.5 with minimum mount of.1 mm KOH. After 15 dys of tretments, for the determintion of the dry weight (DW), plnts were then seprted into roots, stems, nd leves, nd oven-dried t 8 C (for three dys). Besides, fresh shoot nd root smples from eh plnt were immeditely frozen in liquid nitrogen nd stored t 8 C, until performing the iohemil nlysis. Growth nd wter reltions For plnt growth nd ion nlysis, 2 independent dry mtter mesurements nd ion nlysis were performed on seprted leves, stems, nd roots. Shoot or root wter ontent (WC) ws lulted s (FW DW)/FW, where FW nd DW represent the fresh nd dry weight, respetively. The sensitivity index (SI) tht mens the differene etween dry mtter prodution of slt-treted plnts nd the ontrol, expressed in perent of the ltter, were lulted ording to the following expression: SI tretment ¼ð ðdw tretment DW ontrol Þ/DW ontrol Þ. This prmeter SI ws muh lower when the plnt ws sensitive to NCl (Sdllh et l. 21). Ions nlysis For the mesurement of tions, plnt mteril ws dried t 8 C nd digested with nitri id (1% (v/v) HNO 3 ) ording to the method of Wolf (1982). K + nd N + were nlyzed y flme emission using n Eppendorf spetrophotometer. Cl ws quntified y olorimetri method using Digitl Chloridometer HkeBuhler (Buhler instruments In., New Jersey, USA). Free proline nd solule sugrs Proline ontent ws determined using the method of Btes et l. (1973). Proline ws extrted from orgns smples of 2 mg FW with 1 ml of 3% sulphoslyyli id t 7 C for 3 min. After n ddition of id ninhydrin nd glil eti id to the extrts, the mixture ws heted t 9 C for 1 h in wter th. Retion ws then stopped y using n ie th. The mixture ws extrted with toluene, nd the sorne of frtion with toluene spired from the liquid phse were spetrophotometrilly determined t 52 nm. Proline onentrtion ws determined using lirtion urve s μmol proline g 1 FW. Totl solule sugrs were estimted y the nthrone regent method using gluose s the stndrd (Yemm & Willis 1954). Protein extrtion nd quntifition Aliquot of frozen lef nd root mteril ws ground to fine powder with liquid nitrogen nd extrted ( mg FW, 3 ml) t 4 C in mm Tris HCl uffer (ph 8.) ontining 1 mm EDTA, 5 mm KCl, 2 mm MgCl 2,.5 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm dihlorodiphényltrihloroéthne (DDT),.1% (v/ v) Triton X-, nd 1% (w/w) polyvinylpyrrolidone (PVP). The homogente ws entrifuged t 13, g for 4 min t 4 C, nd the superntnt ws lrified y filtrtion. The filtrte ws used for the determintion of ntioxidtive enzyme tivities. Solule protein onentrtion in enzyme extrt ws estimted ording to Brdford (1976), using Sigm regent (B6916) nd ovine serum lumin s stndrd. Three replites per tretment were used.

4 442 A. Mn et l. Ntive gel eletrophoresis nd enzyme tivity stining Polyrylmide gel eletrophoresis (PAGE) ws performed to seprte the different enzyme isoforms using disontinuous gel system under nondenturing onditions, essentilly s desried y Tewri et l. (28). Smples of L. stivum shoot or root extrts were seprted y gel eletrophoresis in 1% (w/v) polyrylmide sl gel t ph 8.9 under ntive onditions, ording to Dvis (1964). Stining for SOD tivity ws rried out s desried y Beuhmp nd Fridovih (1971). The gel ws first soked in 5 mm sodium phosphte, (ph 7.5) ontining 4.8 mm 3-(4,5- dimethylthizol-2-yl)-2,5-diphenyltetrzolium romide (MTT) in drkness for 2 min, followed y soking in 5 mm sodium phosphte (ph 7.5) ontining.4% (v/ v) N,N,N,N-tetrmethylethylenedimine (TEMED) nd 26 mm rioflvin, nd susequently illuminted for 1 min. The three types of SOD, Fe-SOD, Mn-SOD, nd Cu-Zn SOD were identified using inhiitors. Mn- SOD ws visulized y its insensitivity to 5 mm H 2 O 2 nd 2 mm Cynure de potssium (KCN), while Cu-Zn SOD ws sensitive to 2 mm KCN. Fe-SOD ws inhiited y 5 mm H 2 O 2 (Nvri-Izzo et l. 1998). Peroxidse (POD) isoforms were visulized on gels ording to Vllejos (1983). POD ws lolized y inuting the gel for 2 min in retion mixture ontining 2 ml.1 M phosphte uffer (ph 7.), 4 ml.1% H 2 O 2, nd 4 ml.5% guiol or 3,3 -diminoenzidine. It ws then rinsed with distilled wter nd snned immeditely. Sttistil nlysis The sttistil nlyses were performed with the Sttisti softwre (version 6.). All physiologil nd iohemil prmeters, men vlues, nd stndrd error (SE) were otined from 4 to 2 replites nd nlyzed using Dunn s multiple rnge test. A P vlue of <.5 ws onsidered to e sttistilly signifint. For gel nlysis, profile of representtive gel ws reorded y seleting region of interest (lnes) using ImgeJ 1.42 softwre. Results Growth prmeters nd WC We oserved tht 15 dys of slt tretment deresed plnt growth of L. stivum, t ll levels of NCl dded to the medium (Tle 1). The effet of NCl on plnt growth ws ssessed y mesuring the DW of the lef, stem nd root. Fort the whole plnt, redution vlue ofdw, expressed s the rtio of the tretment to the ontrol, ws out 45% under, 15, nd 2 mm NCl (Figure 1A). The mgnitude of the response ws lso linked to the plnt orgn onsidered nd NCl onentrtion. Slt stress tretment induing strong redution of lef, stem, nd root DW t ll levels of NCl exept 5 mm (Figure 1B D). However, the root DW ws more sensitive to slt tretment thn eril prts t onentrtions higher thn 5 mm NCl. Root DW redution ws 38%, 57%, 66%, nd 6%, respetively, for 5,, 15, nd 2 mm NCl (Figure 1D). Although slinity lso indues wter stress omponent in reltion to derese of externl wter potentil, our dt suggest tht s ompred to ontrol tretment NCl tretment hd no effet on lef nd stem WC, exept onentrtion of 2 mm NCl, whih slightly deresed lef WC (18%) nd stem WC (26%) (Figure 2). Chnges in ion umultion L. stivum plnts ultivted under inresing slinity showed signifintly higher N + nd Cl umultion in the leves s ompred to the roots nd stems (Figure 3). The highest onentrtion of N + nd Cl ws reorded with, 15, nd 2 mm NCl. Lef N + onentrtion rnged from. 288 to 49 µmol g 1 DW t 5 mm nd 2 mm NCl, respetively, wheres those of roots vried from. 7 to 26 µmol g 1 DW (Figure 3A). Chloride onentrtion showed similr hnges s sodium (Figure 3B). On the other hnd, slt tretment indued signifint redution of K + ontent s ompred to ontrol tretment (Figure 4A). The mgnitude of K + ontent redution, in leves nd stems, ws stedy whtever the NCl onentrtion. Lef K + ontent vried from. 17 to 6 µmol.g 1 DW t mm nd 2 mm NCl, respetively. However, s ompred to the shoots, root showed the lowest redution of K + ontent t ll NCl onentrtions. This redution ws out 33% s ompred to ontrol tretment (Figure 4A). The osmollity inresed in lef, stem, nd root with inresing slinity. In ft, whtever, the NCl onentrtion supplemented, shoots (leves nd roots) showed the highest osmollity (2*(K + N)) thn the roots nd the medium (Figure 4B). Tle 1. Biomss prodution sensitivity index in L. stivum exposed to slt. NCl (mm) Leves Stems Roots Whole plnt ± ± ± ± ± ± ± ± ± ± ± 2.4 d 52.6 ± ± ± ± ± 1.1 Note: Negtive vlues ( ) orrespond to redution in the dry mtter ompred to ontrol. Dt re mens of 2 replites. Vlues of eh olumn followed y the sme letter indite no signifint differenes (p.5) ording to Dunn s multiple rnge test.

5 Journl of Plnt Intertions 443 Whole plnt dry weight, mg (C) Stem dry weight, mg NCl, mm Lef dry weight, mg Root dry weight, mg 15 5 (D) NCl, mm NCl, mm NCl, mm Figure 1. Effet of NCl tretment on DW prodution of whole plnt, leves, (C) stems, nd (D) roots of L. stivum. Dt re mens of 2 replites ±SE. Mens with similr letters re not different t P <.5 ording to Dunn s multiple rnge test t 95%. Comptile solutes (proline nd sugrs) Totl solule rohydrtes nd proline re the importnt omponents of plnts dpttion to slinity. In the present study, we found n umultion of proline nd totl solule sugrs in L. stivum plnts ultivted under slt onditions (Figure 5). In ft, the proline level grdully inresed with inresing slinity (Figure 5A). Higher levels of proline nd totl solule sugrs were deteted t 2 mm NCl. Whtever the NCl onentrtion, totl solule sugrs nd proline ontents were muh higher in shoots thn roots. Totl solule sugrs level, mrkedly, inresed only in response to 15 nd 2 mm NCl for the shoots (Figure 5B). Shoots sugrs ontent rnged from. 3 to 9 mg.g 1 FW t mm nd 2 mm NCl, respetively, wheres those of roots vried from. 1.7 to 4.2 mg.g 1 FW (Figure 5B). Isozyme ptterns of the ntioxidnt enzymes under NCl stress We explored the impt of NCl on the ntioxidnt isozymes profiles. Indeed, the seprtion of SOD 18 Wter Content ml.g 1 DW 12 6 d d Lef Stem Root Figure 2. Wter ontent in leves, stems, nd root of L. stivum sumitted to NCl tretment (, 5,, 15, nd 2 mm), for 15 dys. Dt re mens of 2 replites ±SE. Mens with similr letters re not different t P <.5 ording to Dunn s multiple rnge test t 95%.

6 444 A. Mn et l N +, µmol/g DW Lef Stem Root Cl -, µmol/g DW Lef Stem Root Figure 3. N + nd Cl ontents of L. stivum exposed to NCl tretment (5,, 15, nd 2 mm). Dt re mens of 2 replites ±SE. Mens with similr letters re not different t P <.5 ording to Dunn s multiple rnge test t 95%. isozymes (fter ntive PAGE) oupled with different speifi inhiitors showed four SOD isozymes in the extrt of lef-solule proteins: one Mn-SOD, two Fe- SODs (denominted Fe-SOD 1 nd Fe-SOD 2 ), nd one CuZn-SOD (Figure 6A). Exmintion of SOD isoenzyme profiles in the roots reveled totl of three isoforms: Mn-SOD, Fe-SOD, nd CuZn-SOD (Figure 6B). Quntifition of the SOD nds intensities y densitometri snning reveled tht Mn-SOD nd Fe- SODs were the predominnt isozymes. The intensity level of ll SOD isozymes inresed under NCl tretments s ompred to ontrol. The highest intensity level of SOD isozymes in leves ws deteted with 5 mm NCl. However, intensity of ll SOD isozymes in roots, grdully, inresed with inresing slinity (Figure 6B). Root Mn-SOD ws more tivted under slinity thn the others SOD isoforms. Ntive PAGE showed four POD isozymes (POD1 4) in the roots nd only one isozyme in the leves (Figure 7). Densitometri snning reveled tht intensity of lef POD isoform, grdully, inresed with inresing slinity nd ws more intense t the slt level of 2 mm (Figure 7A), wheres, slinity tretments hd no effet on root POD isoforms exept onentrtion of NCl 2 mm when the intensity of two POD isozymes (POD1 nd POD2) ws deresed (Figure 7B). Disussion Soil slinity is mjor ioti stress, whih impirs the plnt growth nd development y dversely dmging the vrious plnt metoli proesses, i.e. nitrogen nd sulfur ssimiltion, ntioxidnt system, nd photosynthesis (Zhu 22; Sirm & Tygi 24; Mn et l. 211; Shheen et l. 212). In the present study, we onduted experiments to test the physiologil nd ntioxidnt responses of L. stivum sujeted hydroponilly to slinity stress. Our dt showed tht in the presene of NCl, the growth medium signifintly redued plnt growth of L. stivum. The mgnitude of the response ws lso linked to the plnt orgn onsidered nd NCl onentrtion supplemented to the medium (Figure 1). The present findings gree with those of Muhmmd nd Hussin (21) who report tht slinity, signifintly, inhiited the growth of L. stivum, whih resulted in derese of DW of oth shoots nd roots nd whih ould e ttriuted to the dverse lowering of osmoti potentil. The root growth ws more sensitive to slt tretment thn eril prts (see sensitive index; Tle 1). This sensitivity of the root system seems to e feture of L. stivum. Generlly it is the eril orgns, whih mnifest the depressive effet of slt (Dsgn et l. 22). These results re in greement with the study of Ben Ahmed

7 Journl of Plnt Intertions K +,µmol/g DW Lef Stem Root Lef 2*(K+N) Stem Root Medium Figure 4. K + ontents nd osmollity [2*(K + N)] ofl. stivum exposed to NCl tretment (, 5,, 15, nd 2 mm), for 15 dys. Dt re mens of 2 replites ±SE. Mens with similr letters re not different t P <.5 ording to Dunn s multiple rnge test t 95%. 4 Proline ontent, µmol.g 1 FW Solule sugrs ontent, mg.g 1 FW Shoots e NCl, mm NCl, mm Roots Figure 5. Proline nd sugrs ontents in shoots nd roots of L. stivum exposed to NCl tretment for 15 dys. Dt re mens of 12 replites ±SE. Mens with similr letters re not different t P <.5 ording to Dunn s multiple rnge test t 95%. f d d g f e d et l. (28) onsetri vertiillt. Aording to the uthor, the sensitivity to slinity oserved t the root system of Setri results in derese in iosyntheti tivity. Redution in DW prodution ws ssoited with high umultion of N + nd Cl nd signifint redution of K + ontent in shoots. This limittion in the supply of the plnt in this essentil tion ws oserved in most other Brssiee s Aridopsis thlin (Munns & Tester 28). The growth redution oserved is proly indued y the deline in K + uptke. Indeed, n nlysis of the reltionship etween iomss prodution nd the mount of K + sored in the roots nd exported in shoots (stems nd leves) of plnts sujeted to NCl, showed tht growth of these orgns is highly orrelted with the mount of potssium trnsported nd umulted in them. Although slinity lso indues wter stress omponent in reltion to derese of externl wter potentil, our dt suggest tht under our experimentl onditions, L. stivum ws le to ope with this omponent of slt stress sine no derese in lef WC ws reorded fter 15 dys of exposure to NCl (5,, nd 15 mm). In ontrst, slt-indued inrese in root WC ws even reorded t 5 nd mm NCl (Figure 2), whih might e relted to n ttempt to dilute toxi ions through suulene strtegy. This wter

8 446 A. Mn et l. (NCl, mm) Mn-SOD Fe-SOD 1 Fe-SOD 2 Cu/Zn -SOD 1 (.8) 2 (2.3) 3 (5.3) 4 (1.1) mm 5 mm 1 (13.9) 2 (12.8) 3 (9.9) 4 (3.2) 1 (7.5) 2 (5.8) 3 (4.) 4 (2.3) mm 1 (6.2) 2 (6.) 3 (4.) 4 (1.5) 15 mm 1 (1.9) 2 (7.6) 3 (5.) 4 (2.7) 2 mm (NCl, mm) Mn-SOD Fe-SOD Cu/Zn -SOD 1 (1.3) 2 (7.5) 3 (2.6) mm 1 (1.6) 2 (2.7) 3 (1.8) 5 mm 1 (7.9) 2 (9.8) 3 (5.) mm 1 (18.2) 2 (12.8) 3 (4.9) 15 mm 3 (6.4) 2 mm 1 (3.8) 2 (2.8) Figure 6. Ativity stining of SOD isozymes, in leves nd roots, fter ntive PAGE of NCl-treted L. stivum. Densitometri sns of SOD isozymes from leves nd roots re shown elow the gel imge. The numers indite different isoformi nds. The intensity (vlues in the rket) is expressed in ritrry units. (NCl mm) (3.9) (23.4) (16.2) (17.) (32.2) mm 5 mm mm 15 mm 2 mm (NCl mm) (16.3) 1 (12.2) 1 (18.7) 1 (21.7) 1 (14.6) 2 (27.7) 2 (23.9) 2 (24.5) 2 (28.1) 3 (21.) 2 (11.2) 3 (15.5) 3 (24.3) 3 (21.7) 3 (27.) 4 (15.9) 4 (12.2) 4 (15.) 4 (14.9) 4 (1.3) mm 5 mm mm 15 mm 2 mm Figure 7. Ativity stining of POD isozymes, in leves nd roots fter ntive PAGE of NCl-treted L. stivum. Densitometri sns of POD isozymes from leves nd roots re shown elow the gel imge. The numers indite different isoformi nds. The intensity (vlues in the rket) is expressed in ritrry units.

9 Journl of Plnt Intertions 447 sttus ould e linked to the inrese in osmollity deteted in our experiment. Indeed, the inrese in osmollity hs een regrded s n inditor for osmoti djustment (Frike et l. 24). Under slt stress, plnts hve involved omplex mehnisms llowing for dpttion to osmoti nd ioni stress used y high slinity. These mehnisms inludes osmoti djustment y umultion of omptile solutes suh s proline nd sugrs (Gill & Tutej 21). Amino id proline, whih is known to our widely in higher plnts, normlly umultes in lrge mounts in response to vrious ioti stresses (Ashrf 1994; Ali et l. 1999). Proline ws known to e n osmoregultory ompound involved in mintining the wter lne of the plnt exposed to slinity (Okum et l. 24). It not only ts s ytoplsmi osmolyte filitting wter retention ut lso s protetor nd stilizer of mromoleules nd ellulr strutures (Bohnert & Jensen 1996). It hs long een suggested tht umultion of proline in plnt tissue under slt stress is n dpttive response even though investigtors hve otined ontrsting results regrding the role of proline in stress tolerne of plnts (Ashrf 1994; Rhodes et l. 1999). In L. stivum, the proline ontent in shoots nd roots ws gretly inresed fter 15 dys of tretment in the presene of NCl. The umultion of this solute in the different orgns is proportionl to the mount of slt dded to the medium. On the other hnd, sugrs re onsidered to ply mjor role in osmoregultion under ioti stress onditions (Fllon & Phillips 1988; Ptde et l. 211). In ft, the first effet of slinity on plnts is drought effet or wter defiit. Plnts try to redue their osmoti potentil vi inresing minerl ions ontent nd omptile solutes synthesis to etter wter uptke under slinity. Totl solule rohydrtes re importnt solutes tht re synthesized nd umulted in ytosol under slt stress. Thus, they re neessry for the survivl of the plnt euse they re soure of ron nd energy (Rejskov et l. 27; Krsensky & Jonk 212). In our study, the presene of NCl in the medium lso indues n inrese in totl solule sugrs ontents. Colletively, the signifint umultion of proline nd sugrs in the different orgns ws proly ssoited with osmoti djustment in L. stivum. Exposure of plnts to slt stress inrese the prodution of ROS suh s singlet oxygen ( 1 O 2 ), superoxide rdil (O 2 ), hydrogen peroxide (H 2 O 2 ), nd hydroxyl rdil (OH ), nd these ROS use oxidtive dmge to different ellulr omponents, inluding memrne lipids, protein, nd nulei ids (Apel & Hirt 24; Tnk et l. 26). Previous dt reveled tht the lne of ROS formtion nd the removl re determinnt ftor for the severity of oxidtive stress nd ell dmge (Mittler et l. 24; Shi et l. 26). Previous works showed tht slt tolerne is losely relted to the effiieny of ntioxidnt enzymes (Khn et l. 29; Shheen et l. 212). SOD nd POD re mong the mjor ntioxidnt enzymes involved in svenging ROS (Askri et l. 26; Bhushn et l. 27). Our dt support this hypothesis sine lerly displying signifintly inresed SOD nd POD isoforms tivities in shoots nd roots of L. stivum plnts ultivted under inresing slinity. Our results ontrst with previous work whih reveled tht slt indued ltertions in the ntioxidtive system (Sleh & Plieth 29). Aording to the uthor, slt stress (15 mm NCl) inhiits the glutthione redutse (GR) nd CAT of L. stivum seedlings. On the sis of these results, it ws onluded tht L. stivum n e lssified s modertely tolernt plnt to slt stress. The slt tolerne of L. stivum would e prtilly sed on: its ility to mintin onvenient tissue wter supply, osmoti djustment y umultion of osmoregultory ompounds suh s proline nd sugrs, the ptitude of the whole plnt to ensure suffiient K + supply y mintining high seletivity for this essentil nutrient in spite of lrge mount of N + in the medium nd to exhiit high-ntioxidnt enzyme tivities (SOD POD), preventing the toxi umultion of ROS. Aknowledgment The uthors knowledge the Ministry of Higher Edution nd Sientifi Reserh of Tunisi for finnil support. Mny thnks to Mr. Fdhel BEN AICH for tehnil ssistne nd tking re of the plnts. We lso thnk Dr. Adellh CHALH for sttistil help nd Amel Terrs ritil reding of the mnusript. Referenes Ali G, Srivstv PS, Iql M Proline umultion, protein pttern nd photosynthesis in Bop monnier regenernts grown under NCl stress. Biologi Plnt. 42: Apel K, Hirt H. 24. Retive oxygen speies: metolism, oxidtive stress, nd signl trnsdution. 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