Is salinity tolerance related to osmolytes accumulation in Lygeum spartum L. seedlings?

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1 Journl of the Sudi Soiety of Agriulturl Sienes (2011) 10, King Sud University Journl of the Sudi Soiety of Agriulturl Sienes ORIGINAL ARTICLE Is slinity tolerne relted to osmolytes umultion in Lygeum sprtum L. seedlings? Bouzid Nedjimi * Lortoire d Explortion et Vloristion des Eosyste`mes Steppiques, Universite de Djelf, Fulte des Sienes de l Nture et de l Vie, Cite Aıˆn Chih, BP 3117, Djelf 17000, Algeri Reeived 22 Ferury 2011; epted 22 Mrh 2011 Aville online 17 My 2011 KEYWORDS Free mino ids; HPLC; Lygeum sprtum L.; NCl; Osmoti djustment; Totl solule sugrs Astrt Lygeum sprtum L. (Poee) is plnt of ommeril relevne used s rw mteril for mnufturing pper. This speies is newly found slt tolernt speies, ut its physiologil responses to slinity re poorly understood. The effet of slt stress (50 nd 100 mm NCl) on growth, lef wter reltions, solule sugrs nd free mino ids in L. sprtum hs een investigted. Fresh nd dry weights were redued signifintly ove 50 mm NCl. Trnspirtion, wter potentil (W x ) nd osmoti potentil (W p ) deresed with elevted NCl. No hnge ws oserved in the turgor potentil (W s ). Susequently, the omposition of free mino ids estimted y high pressure liquid hromtogrphy (HPLC) indited signifint inrese in free mino id ontent. It ppers tht vline ws the min mino id umulted signifintly y the plnts for oth NCl tretments. However, tyrosine levels derese y slt tretment ompred to ontrol. Contents of N + nd Cl inresed with n inrese in slinity. The onentrtion of N + of slinized plnts (100 mm NCl) ws 70-fold greter thn tht mesured in ontrol plnts, nd this ws ssoited with signifint redutions in lef K + nd C 2+ onentrtions. In ddition, signifint umultion of solule sugrs, proly ssoited with osmoti djustment nd protetion of Arevitions: HPLC, high pressure liquid hromtogrphy; W p, osmoti potentil; W s, turgor potentil; W x, wter potentil. * Tel.: ; fx: E-mil ddress: nedjimi@yhoo.fr X ª 2011 King Sud University. Prodution nd hosting y Elsevier B.V. All rights reserved. Peer review under responsiility of King Sud University. doi: /j.jsss Prodution nd hosting y Elsevier

2 82 B. Nedjimi memrne stility, ourred in roots of slinized plnts. Bsed upon these results, possile physiologil role of solule sugrs nd free mino ids ws suggested in L. sprtum to mintin turgor. ª 2011 King Sud University. Prodution nd hosting y Elsevier B.V. All rights reserved. 1. Introdution Slinity is n importnt ftor limiting griulturl produtivity in rid nd semirid regions of Algeri (Nedjimi nd Doud, 2009). Reliming these lnds for forge rops is too ostly for most ountries to fford. A delining se of rle frmlnd nd n inresing demnd for forge nd fire wrrnt the need for utiliztion of the nturlly slt tolernt speies in irrigted nd non-irrigted griulture (Reddy et l., 2008). Plnts exposed to slt stress undergo hnges in their environment. The ility of plnts to tolerte slt is determined y the multiple iohemil pthwys tht filitte retention nd/or quisition of wter, protet hloroplst funtions, nd mintin ion homeostsis. Essentil pthwys inlude those tht led to synthesis of osmotilly tive metolites, speifi proteins, nd ertin free rdil svenging enzymes tht ontrol ion nd wter flux nd support svenging of oxygen rdils (Prid nd Ds, 2005). Mny plnt speies respond rpidly to stressors y inresing the onentrtion of omptile solutes involved in osmoregultion nd in protetion of proteins nd memrnes in onditions of low wter potentil (Munns nd Tester, 2008). The diret domestition of slt tolernt plnts represents n lterntive pproh to expnd ultivtion onto unfvourle lnd, nd it n e envisioned s strtegy omplementry to the geneti engineering of slt tolerne in glyophytes. Therefore, for most speies, their potentil eonomi use s ultivted plnts nd their dptility to gronomi onditions hve not een fully ssessed. Lygeum sprtum L. (Poee) is ntive speies in the Algerin slt steppes (Fig. 1). The plnt is of interest euse of its tolerne to environmentl stresses nd its use s fodder grss for livestok in low-rinfll Mediterrnen res (Nedjimi, 2009). This speies n tolerte extreme onditions of ridity, slinity nd high tempertures (Le Houe rou, 1995). Its extensive root system plys signifint role in preventing desertifition y stilizing the snd (Pugnire nd Hse, Figure 1 Lygeum sprtum L. (Poee). 1996). It my thus e suitle ndidte for the phytostiliztion of id mine tilings (Cones et l., 2007). It hs een used s fodder for domesti livestok nd for rehilittion of degrded lnds (gypsum nd lreous soils) (Gri- Fuentes et l., 2001). In North Afri, it ws used s rw mteril for mnufturing pper (FAO, 1992). L. sprtum dpts very well to rid nd semi-rid soils nd shows high growth pity under these onditions (Nedjimi, 2009; Nedjimi et l., 2010). However, physiologil response of L. sprtum to slt stress is not well understood lthough slinity is often ssoited with the environment where it is grown. In previous works, Nedjimi (2009) demonstrted L. sprtum s modertely tolernt to slinity. Until now, suh omprehensive investigtion on L. sprtum possile omptile solutes hs not een done. The hypotheses tested were (1) tht free mino id umultes in stressed L. sprtum to high levels whih n e importnt for osmoti dpttion, nd (2) tht slinity ws le to indue solule sugr umultion in this speies. The study ims to dd rguments in fvour of signifint ontriution of osmolytes to the slinity tolerne of L. sprtum. 2. Mterils nd methods 2.1. Plnt mteril nd growth onditions The seeds of L. sprtum were olleted from the re of Aı n Mˆed in the provine of Djelf (Algeri) ( E longitude, N ltitude nd 934 m elevtion). Seeds were pre-hydrted with erted, de-ionised wter for 12 h nd germinted in vermiulite, t 28 C in n inutor, for 2 d. They were then trnsferred to ontrolled-environment hmer with 16 h light 8 h drk yle nd ir tempertures of 25 nd 20 C, respetively. The reltive humidity (RH) ws 60% (dy) nd 80% (night) nd photosynthetilly tive rdition (PAR) ws 400 lmol m 2 s 1, provided y omintion of fluoresent tues (Philips TLD 36 W/83, Germny nd Sylvni F36 W/GRO, USA) nd metl hlide lmps (Osrm HQI.T 400 W, Germny). After 7 d, the seedlings were pled in 15-L ontiners (six plnt per ontiner) with ontinuously erted, modified Hoglnd nutrient solution (Hoglnd nd Arnon, 1938): C(NO 3 ) 2 (2 mm), K 2 HPO 4 (0.5 mm), MgSO 4 (0.5 mm), H 3 BO 3 (25 lm), MnSO 4 (2 lm), ZnSO 4 (2 lm), CuSO 4 (0.5 lm), (NH 4 ) 6 Mo 7 O 24 (0.5 lm), Fe-EDDHA [Fe-ethylendimino-di(o-hydroxyphenyleti) id] (20 lm). The solution ws repled ompletely every week. After 13 d (when plnts were 20 d-old), plnts were treted with 0, 50 nd 100 mm NCl. The experiment ws set up s Completely Rndomized Design. Eh tretment ws replited five times nd eh replite inluded six plnts (i.e., 30 plnts per tretment). Dry nd fresh weights, wter potentil (W x ), osmoti potentil (W p ), turgor potentil (W s ) nd mino ids were mesured fter 30 d of the tretments, when plnts were 50 d.

3 Osmolytes umultion in Lygeum sprtum L Mesurement of fresh nd dry weights Plnts were hrvested nd totl fresh weight (FW) ws determined. The totl dry weight (DW) ws mesured fter the smples hd een dried t 65 C for 72 h Plnt trnspirtion For plnt trnspirtion mesurement, eh pot ontining one plnt ws overed with plsti g, seured round the stem se. The wter trnspired ws estimted under ontrolled light from the weight loss over 6-h period (10:00 16:00 h) orresponding to high nturl sunlight period. The men trnspirtion rte (per g FW) ws lulted sed on the mount of trnspired wter nd totl fresh weight t smpling time Lef wter reltions The lef wter potentil (W x ) of the most reent fully-expnded leves ws mesured using the pressure hmer tehnique (Turner, 1988). The sme leves were put into plsti gs nd rpidly frozen with liquid nitrogen. They were susequently thwed nd pressed to extrt the ell sp. The osmoti potentil (W p ) of the lef sp ws lulted fter mesuring sp osmolrity using n utomti, freezing-point depression osmometer (Digitl Osmometer, Roeling, Berlin), y vn t Hoff eqution (Noel, 1991): Wp ¼ nrt: where n = mosmol, R= nd T=t mient temperture (K). Turgor potentil (W s ) ws lulted s the differene etween lef wter potentil nd osmoti potentil Chemil nlysis Leves smples were put in Eppendorf tues with holes t the ottom nd rpidly frozen with liquid nitrogen. These tues were then entrifuged twie into ssy tues, t 4000 g for 4 min (4 C), in suh wy tht ll the sp ws extrted from the smples. For the ion nlysis, 25 ll of ell sp ws filtered, diluted nd injeted into Dionex-D-100 ion hromtogrph with n Ionp AS124-4 mm (10 32) olumn nd n AG 14 (4 50 mm) gurd olumn. Chloride (Cl ) ws mesured with Chromeleon/Peknet 6.40 hromtogrphy softwre, y ompring pek res with those of known stndrds. The sodium (N + ), potssium (K + ) nd lium (C 2+ ) onentrtion in the ell sp, determined y tomi sorption spetrometry (905AA, GBC, Austrli), ws mesured for extrt liquots diluted with LCl 3 + CsCl solution Free mino ids nlysis The nlyses were rried out with n HPLC/MS system onsisting of n Agilent 1100 series HPLC (Agilent Tehnologies, Snt Clr, CA, USA), l-well plte uto-smpler nd pillry pump, onneted to n Agilent Ion Trp XCT Plus Mss Spetrometer (Agilent Tehnologies) using n eletrospry (ESI) interfe. Stndrds, with known onentrtions of eh mino id (0.1, 0.5, 1, 10, 25 nd 50 lm), nd smples were prepred in the moile phse A, onsisting of wter/etonitrile/formi id (89.9:10:0.1), nd pssed through 0.22-lm filters. Then, 5 ll of eh stndrd or smple ws injeted onto Zorx SB-C18 HPLC olumn (5 lm, mm, Agilent Tehnologies), thermosttted t 40 C, nd eluted t flow rte of 5 ll min 1. The moile phse B, onsisting of wter/etonitrile/formi id (10:89.9:0.1), ws used for the hromtogrphi seprtion. The elution onsisted of 5 min of 0% B, liner grdient from 0% to 10% B in 10 min nd 10% B for 5 min. The olumn ws equilirted with the strting omposition of the moile phse for 20 min efore eh nlytil run. The UV hromtogrm ws reorded t 210 nm with the DAD module (Agilent Tehnologies). The mss spetrometer ws operted in the positive mode, with pillry spry voltge of 3500 V nd sn speed of 26,000 (mz 1 )s 1 from 50 to 250 mz 1. The neuliser gs (He) pressure ws set to 15 psi nd the drying gs ws set to flow of 5 l min 1, t 350 C. The hromtogrm of eh mino idi ion from oth stndrds nd smples ws extrted nd the pek re ws quntified using the Dt Anlysis progrmme for LC/MSD Trp Version 3.2 (Bruker Dltonik, GmH, Germny). The pek re dt of the stndrds were used for lultion of the lirtion urve, from whih the onentrtions of eh mino id in the smples were otined Solule sugrs nlysis Totl solule sugrs ontent in lef sp ws mesured ording to the phenol sulfuri id of Duios et l. (1956) method Dt nlysis Dt were nlysed sttistilly, using the SPSS 7.5 softwre pkge, y ANOVA nd y Tukey s multiple rnge test, to determine differenes etween mens. 3. Results Fresh weight (P < 0.001) nd dry weight (P < 0.01) of L. sprtum plnts were ffeted y slinity. Growth prmeters mentioned ove deresed signifintly t high NCl onentrtion. These prmeters did not hnge signifintly t 50 mm NCl (Tle 1). In ddition, trnspirtion in L. sprtum L. seedlings delined signifintly (P < 0.01) with the inrese of slinity (Tle 1). Anlysis of leve sp reveled tht the wter potentil (W x ) (P < 0.001) nd osmoti potentil (W p )(P < 0.01) were deresed y the high-ncl tretment (100 mm NCl) (Fig. 2). However, there were no signifint differenes (P > 0.05) etween the turgor potentil (W s ) vlues of the ontrol nd treted plnts (Fig. 2). Slinity signifintly ffeted N + (P < ), K + (P < ), C 2+ (P < ), nd Cl (P < 0.005) ontents of plnts. Contents of N + nd Cl inresed with n inrese in slinity. The onentrtion of N + in leves sp of plnts treted with 100 mm NCl ws 70-fold greter thn tht mesured in ontrol plnts. Seedlings lso umulted signifintly higher Cl thn their orresponding ontrols (Fig. 3). The lium (C 2+ ) nd potssium (K + ) ontents of plnts deresed with n inrese in slinity (Fig. 3).

4 84 B. Nedjimi Tle 1 Effets of NCl on FW nd DW, trnspirtion nd of Lygeum sprtum L. grown in hydroponi onditions. Vlues represent mens ± stndrd error (n = 5). NCl (mm) FW (g plnt 1 ) DW (g plnt 1 ) Trnspirtion (g H 2 O g plnt 1 ) ± ± ± ± ± ± ± ± ± 0.05 Different letters in the sme olumn indite signifint differene t the 5% level ording to the Tukey s multiple rnge test r²= Vline Asprgine Phenyllnine Tyrosine Potentiels (MP) NCl (mm) Ψ ω (MP) Ψπ (MP) Ψτ (MP) r²=0.92 r²=0.78 Amino ids (μm) NCl (mm) Figure 2 Effets of NCl on wter potentil (W x ), osmoti potentil (W p ) nd turgor potentil (W s )oflygeum sprtum L. grown in hydroponi onditions. Dt represent mens ± SE (n = 5). Vlues with different letters re signifintly different (P < 0.01, Tukey s test). Figure 4 Effets of NCl on mino ids ontent of Lygeum sprtum L. grown in hydroponi onditions. Dt represent mens ± SE (n = 5). Vlues with different letters re signifintly different (P < 0.01, Tukey s test). Ions (mm) Sodium Potssium Clium Chloride Totl solules sugrs (mm) NCl (mm) 0 NCl (mm) Figure 3 Effets of NCl on N +,K +,C +2 nd Cl ontents of Lygeum sprtum L. grown in hydroponi onditions. Dt represent mens ± SE (n = 5). Vlues with different letters re signifintly different (P < 0.01, Tukey s test). Amino ids were lso nlysed in the extrt ell sp. When individul mino ids were studied, only the mounts of some of them hnged s onsequene of the tretments (Fig. 4). Vline ws mjor mino id inresed signifintly for oth NCl tretments. An inrese in phenyllnine ws Figure 5 Effets of NCl on totl solule sugrs ontent of Lygeum sprtum L. grown in hydroponi onditions. lso oserved for oth tretments, ompred with ontrol plnts. However, generl nd similr derese in tyrosine levels ourred in the sline tretments ompred to ontrol (Fig. 4). After 30 d of tretments, slinity hd signifint effet on totl solule sugrs ontent in L. sprtum plnts (P < 0.05).

5 Osmolytes umultion in Lygeum sprtum L. 85 Totl solule sugrs ontent sustntilly inresed with n inrese in slinity (Fig. 5). 4. Disussion Totl mino ids (mm) NCl (mm) Figure 6 Effets of NCl on totl mino ids ontent of Lygeum sprtum L. grown in hydroponi onditions. Dt represent mens ± SE (n = 5). Vlues with different letters re signifintly different (P < 0.01, Tukey s test). The present study showed tht FW nd DW of L. sprtum L. were deresed signifintly with the inrese in NCl tretments (Tle 1). Similr results hve een reported for other grsses (Bi et l., 2008; Gulzr nd Khn, 2006; Musolo et l., 2003). Thus, the growth redution oserved in plnts sujeted to slt stress often results from diret effets (toxiity of ions umulted in tissues) nd/or from indiret effets (limittion of minerl nd wter quisition) (Nedjimi nd Doud, 2009; Nedjimi, 2009). Trnspirtion rtes of our plnts deresed signifintly with the inrese of slinity (Tle 1). This my e due to the possiility tht lowered wter potentils in the roots n trigger signl from root to shoot. This signl might e hydrostti or involve orgni moleules suh s ABA, s previous uthors hve limed (Zhng nd Dvies, 1991). Osmoti djustment involves the net umultion of solutes in ells in response to fll in the wter potentil of their environment. As onsequene of this net umultion, the ell osmoti potentil is lowered nd turgor pressure tends to e mintined (Mrum nd Murdoh, 1992). The redutions oserved in osmoti potentil (W p ), when tretments were pplied, were well relted to redutions in wter potentil (W x ), ut hd no effet on turgor (Fig. 2). The ft tht there were no hnges in turgor in the plnts, in response to the tretments of our experiment, indites ertin level of osmoti djustment (Munns, 2002; Nedjimi et l., 2010). The presene of high onentrtions of N + nd Cl in the nutrient solution produed high uptke of these ions nd ontriuted to their inresed flux into the xylem (Fig. 3), suggesting tht these were the inorgni solutes involved in osmoti djustment (Munns nd Tester, 2008). This effet hs een reported in other Poee s well, e.g., in Sporoolus virginius (Mrum nd Murdoh, 1992), Oryz stiv (Alm et l., 2002), Hordeum vulgre (Shl et l., 2005) nd Iris lte (Bi et l., 2008).The redution of growth indued y slinity (Tle 1) ws proly ssoited with the toxi effet of the umultion of N + nd Cl in plnt tissues nd redution of sorption of K + nd C 2+. High N + onentrtion interferes with intrellulr K + nd C 2+ umultion presumly y ompeting for the sme sites of influx (Tester nd Dvenport, Therefore, the key for tolerne might e synhronistion etween the high rte of ion trnsport to the shoot nd ion omprtmenttion y the lef ells (Munns et l., 2006). In our experiments, L. sprtum umulted lrge mount of N + nd Cl ions nd lower mount of C 2+ nd K +. First the similr physiohemil strutures of N + nd K + men tht N + ompetition t trnsport sites for K + entry into the symplst my result in K + defiieny. Seondly, ytoplsmi N + ompetes for K + inding sites nd hene inhiits metoli proesses tht ruilly depend on K + (Mthius nd Amtmnn, 1999). Under sline onditions, high levels of externl N + not only interfere with C 2+ quisition y the roots, ut lso my disrupt the integrity of root memrnes nd lter their seletivity (Grttn nd Grieve, 1999). Sine mintining n dequte supply of C 2+ in sline soil solutions is n importnt ftor in ontrolling the severity of speifi ion toxiities, prtiulrly in rops whih re suseptile to N + nd Cl injury (Nedjimi nd Doud, 2009). Osmoti djustment, whih is neessry for growth in sline environment, my e omplished y umultion of inorgni nd orgni solutes. Inorgni ions re elieved to e sequestered in the vuoles, while orgni solutes re ssumed to e omprtmentlised in the ytoplsm to lne the low osmoti potentil in the vuole (Munns nd Tester, 2008). Mssive umultion of free mino ids under slt stress hs een reported in mny grmineous speies (Lutts et l., 1999; Mornt-Mneu et l., 2004; Wng et l., 2007). In the present experiment with L. sprtum, slinity inresed the totl mino ids, ompred with the ontrol plnts (Fig. 6). However, when individul mino ids were studied, only n inrese in lef sp vline onentrtions, to vlues nerly doule those oserved in ontrol plnts were reported (Fig. 5). The inrese of vline ws positively relted to Cl (r = 0.93, dt not shown) nd N + (r = 0.85, dt not shown) umultions. Amino ids hve een reported to umulte in higher plnts under slinity stress (Ashrf, 1994). The importnt mino ids inlude lnine, rginine, sprgines, glyine, serine, nd leuine, together with the mino id, proline, nd the non-protein mino ids, itrulline nd ornithine (Ashrf nd Hrris, 2004). Vline ws lso een reported to umulte in plnts in response to slt stress suh s strwerry (Keutgen nd Pwelzik, 2008), Bet vulgris (Gzik, 1996) nd Phrgmites ustrlis (Hrtzendorf nd Rolletshek, 2001). Free mino id umultion in plnts under slt stress hs often een ttriuted to ltertions in iosynthesis nd degrdtion proesses of mino ids nd proteins (Hre et l., 1998). Considering tht slinity inresed the free mino id ontent in sp leves (Fig. 6), our results ould e relted to n inrese in mino id degrdtion or inhiition in synthesis jointly with redutions in degrdtion or inreses in protein synthesis. In this work, the ontriution of totl solule sugrs umultion to osmoti djustment ws signifint, sine the totl

6 86 B. Nedjimi solule sugrs ontent inresed with n inrese in slinity (Fig. 5). Similr results were otined y Mornt-Mneu et l. (2004), who reported tht the onentrtions of sugrs hnge in response to slt stress in Tritium dioum. Crohydrtes suh s solule sugrs (gluose, frutose, surose, frutns) umulte under slt stress to ommodte the ioni lne in the vuoles (Prid nd Ds, 2005; Ashrf nd Hrris, 2004). Their mjor funtions re osmoprotetion, osmoti djustment, ron storge, rdil svenging nd stiliztion of the struture of proteins suh s Ruiso (Rejsikov et l., 2007). The tivity of surose phosphte synthse inreses under slt stress, wheres strh phosphorylse tivity dereses (Duey nd Singh, 1999). Solule sugr umultion my e due to further trnsformtion of strh to sugrs or less onsumption of rohydrtes y the tissues in sline onditions (Hre et l., 1998). To onlude, the strtegies of slt tolerne in L. sprtum involve delite lne mong ion umultion, osmoti djustment, solule sugrs nd mino ids prodution nd mintenne of pressure potentil. Further studies on enzymti nd growth hormones re required to eluidte the iohemil mehnisms implied. Aknowledgement This reserh ws finnilly supported y the Algerin Ministry of Higher Edution nd Sientifi Reserh (Projet no. F ). Referenes Alm, S., Immul Huq, S.M., Kwi, S., Islm, A., Effets of pplying lium slts to ostl sline soils on growth nd minerl nutrition of rie vrieties. Journl of Plnt Nutrition 25, Ashrf, M., Orgni sustnes responsile for slt tolerne in Eru stiv. Biologi Plntrum 36, Ashrf, M., Hrris, P.J.C., Potentil iohemil inditors of slinity tolerne in plnts. Plnt Siene 166, Bi, W.B., Li, P.F., Li, B.G., Fujiym, H., Fn, F.C., Some physiologil responses of Chinese Iris to slt stress. 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