Environmental and Experimental Botany

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1 Environmentl nd Experimentl otny 87 (213) ontents lists ville t SiVerse SieneDiret Environmentl nd Experimentl otny journ l h omep g e: Slt tolerne of enture rgusin L. is ssoited with effiient osmoti djustment nd inresed ntioxidtive pity Sndr Rdić,, Petr Pehre Štefnić, Hrvoje Lepeduš, Vior Roje, rnk Pevlek-Kozlin University of Zgre, Fulty of Siene, Deprtment of iology, Rooseveltov trg 6/III, HR-1 Zgre, roti griulturl Institute Osijek, Južno predgrd e 17, HR-31 Osijek, roti, 1, Zgre, roti University of Zgre, Fulty of Forestry, Svetošimunsk 25, HR-1, Zgre, roti r t i l e i n f o rtile history: Reeived 18 June 212 Reeived in revised form 22 Otoer 212 epted 1 Novemer 212 Keywords: Slinity Suulene Photosystem II ntioxidnt ronyl Lipoxygense s u m m r y The present study investigted the effets of slinity on the perennil speies enture rgusin L. interesting s potentil sh rop plnt. Plnts grown in ulture onditions were sujeted to inresing slt ( 6 mm Nl) or mnnitol (3 mm) tretments for two weeks. Effets of isoosmoti onentrtions of Nl (15 mm) nd mnnitol were ompred nd disussed in order to disriminte possile differenes in. rgusin response to ioni (Nl) nd osmoti (mnnitol) omponents of slinity.. rgusin plnts used N nd to lesser extent l ions s primry osmoti though with higher slinity, proline umultion inresed s well. onurrently, with inresing slinity signifint redutions in plnt K, Mg nd onentrtions ourred. In ddition, lower slt onentrtions indued lef suulene nd inresed lef reltive wter ontent (RW). threshold slinity ove whih.rgusin L. showed signs of dmge nd growth inhiition ws rehed t 3 mm. tivities of superoxide dismutse, tlse nd sorte peroxidse in slinized plnts seem to ply n essentil protetive role in the svenging proesses. Regrdless of the high indution of ntioxidtive system nd mssive proline umultion, mnnitol used derese of RW nd oxidtive dmge to proteins nd lipids. onsidering the undne of some photosyntheti proteins (Ruiso, D1, LHI, LHII nd FNR) nd PSII effiieny, it n e onluded tht oth slt nd mnnitol impired photosynthesis in. rgusin though slt to muh lesser extent. The results suggest tht the mjor reson for the prtiulr threshold of slinity tolerne in. rgusin n e ttriuted to limited dilution pity of suulent tissue. The tolerne strtegies of. rgusin to moderte slinity seem to inlude osmoti djustment hieved through slt ions uptke s dominnt strtegy ut lso highly induile ntioxidtive defense. 212 Elsevier.V. ll rights reserved. 1. Introdution Slinity n inhiit plnt growth due to vrious ftors, inluding ion toxiity, hnges in the wter reltions, impirment of minerl nutrition nd intivtion of photosyntheti mhinery. The extent to whih eh of these ftors n ffet growth depends on dpttions to oth low wter potentils nd high sodium onentrtions (Munns, 22). Slt-indued osmoti nd ioni stress distur the ellulr redox lne using over redution of photosyntheti eletron trnsport hin nd thus mplifying prodution revitions: FNR, Ferredoxin (Fd): NDPH oxidoredutse; LHI, light hrvesting omplex of photosystem I; LHII, light hrvesting omplex of photosystem II; PSII, photosystem II; Ruiso, lrge suunit of riulose-1,5-isphosphte roxylse oxygense. orresponding uthor. Tel.: ; fx: E-mil ddresses: sndr@zg.iol.pmf.hr (S. Rdić), ppehre@zg.iol.pmf.hr (P. Pehre Štefnić), hlepedus@yhoo.om (H. Lepeduš), vroje@sumfk.hr (V. Roje). of retive oxygen speies (ROS). Some of the exessive energy not utilized in photohemistry is quenhed into hlorophyll fluoresene to minimize dmge to photosystems, prtiulrly in PSII nd susequent eletron rriers (Kruse nd Weis, 1991). The highly retive ROS re ytotoxi when overly produed nd n dmge lipids, proteins, nulei ids nd photosyntheti omponents. Polyunsturted ftty ids of plsm memrne re mong the more suseptile iologil moleules to oxidtion. They n esily e oxidized into their orresponding hydroperoxides y ROS or enzymtilly y lipoxygense (LOX, E ). Detoxifition of ROS in plnts is ontrolled y enzymti defense systems suh s superoxide dismutse (SOD, E ), tlse (T, E ), vriety of peroxidses (sorte peroxidse PX, E , guiol peroxidse GPX, E ) nd nonenzymti ones of whih sorte is the most undnt (Prid nd Ds, 25). The ility of plnts to survive nd mintin their growth under sline onditions is known s slt tolerne. In summry, mehnisms of slt tolerne re of two min types: those minimizing the /$ see front mtter 212 Elsevier.V. ll rights reserved.

2 4 S. Rdić et l. / Environmentl nd Experimentl otny 87 (213) entry of slt into the plnt (or t lest their umultion in photosyntheti tissues) nd those minimizing the onentrtion of slt in the ytoplsm (Munns, 22). This orresponds with two mjor dptive strtegies of plnts to tolerte high environmentl slinity: 1) stress voidne, relted to different physil, physiologil nd/or metoli rriers with whih the dverse effets of stress re meliorted, nd 2) stress tolerne, relted to dptive mehnisms whih enle suessful survivl despite the influene of stress internlly. Slt tolernt speies re often le to umulte high onentrtions of slts in their tissues for osmoti djustment through the omprtmentliztion of ions in vuoles nd the prodution of omptile solutes suh s proline in the ytoplsm (Prid nd Ds, 25). Proline my lso protet enzymes (proteins) from oxidtive dmge under slinity or dehydrtion stress (Ghoulm et l., 22). Erlier studies hve suggested tht tolerne of plnts to slt stress is ssoited with the indution of ntioxidnt enzymes (or et l., 23; en mor et l., 25). enture rgusin L. is rotin perennil plnt whih is, like some other speies in enture genus (Pieroni et l., 22; Rusk et l., 22; rif et l., 24; N et l., 28) interesting s potentil sh rop plnt due to numer of iotive phytohemils with potentil mediinl or phrmeutil pplitions (unpulished dt). It thrives long the vertil limestone liff-fes of its ntive hitt ost of the driti Se nd on some islnds nd is thus simultneously ffeted y high light irrdine, drought nd slt. ordingly, it hs een desried s hlophyte with ertin xeromorphi hrteristis, suh s very dense gry-white hir over, thik utile nd plisde prenhym more developed thn spongy prenhym, whih serves s wter storing tissue (čić et l., 1997). However, the tolerne of tht plnt speies to slinity hs yet to e estimted. The oservtion of slt indued suulene (Rdić et l., 26) implied tht. rgusin hs no exlusion mehnism t root level to void exess umultion of slt ions in leves. Suulene is onsidered s n dpttion tolerne hrteristi of hlophytes in terms of onservtion of internl wter, effiient wter storge nd dilution of umulted slts (Flowers et l., 1986; Wng et l., 212). sed on the previous results (Rdić et l., 26) nd regrding the nturl hitt of. rgusin, we ssumed its tolerne to slinity s well s to oxidtive stress. In this study we imed to lrify physiologil strtegies leding to the slinity tolerne of this speies. Moreover, we disuss differenes in. rgusin dpttion mehnisms in response to slt or mnnitol stress y ompring ertin morpho-physiologil nd iohemil prmeters lef ntomy, ion distriution, proline ontent, photosystem II effiieny, levels of some photosyntheti proteins (Ruiso LSU, D1, LHI, LHII nd FNR), nd ertin detoxifying enzymes nd ntioxidnts. In our previous investigtion, gret umultion of H 2 O 2 in mnnitol-treted. rgusin ws notied while slt did not ffet the level of tht oxygen speies fter 2-week period (Rdić et l., 26). Here, the tivity of lipoxygense, the hydroperoxide generting enzyme, nd ronyl groups ontent, n inditor of oxidtive dmge to proteins, were lso investigted. 2. Mteril nd methods 2.1. Plnt mteril nd ulture onditions. rgusin seeds were olleted from their nturl hitt ner Durovnik (rok formtion Konvoske stijene lolity Psjč). The sterilized seeds were germinted in ontiners filled with MS ½ medium ontining.1 g L 1 myo-inositol,.1 mg L 1 thimine Hl,.5 mg L 1 pyridoxine Hl,.5 mg L 1 niotini id, 2.9 mm gierelli id (G3),.5 mm 6-enzylminopurine (), 3 g L 1 surose nd 8 g L 1 gr (Murshige nd Skoog, 1962). Four-week old plnts were suultured to liquid MS ½ medium nd, following root initition, were trnsferred to the sme omposition medi supplemented with 15 (17.51 ms m 1,), 3 (3.4 ms m 1,), 45 (44.1 ms m 1,) nd 6 mm (55. ms m 1,) Nl or 3 mm mnnitol (2.38 ms m 1,) orresponding to osmoti potentils.85, 1.5, 2.44, 3. or.84 MP, respetively. ontrol (2.52 ms m 1,.14 MP) plnts were kept in nutrient solution without slt or mnnitol. ll nlyses were performed fter 15 dys of further growth. The plnts were grown in growth hmer t 24 ± 2 under 16:8 h light:drk period of ool fluoresent light (9 E m 2 s 1 ) Reltive growth rte, reltive wter ontent nd proline ontent Reltive growth rte (RGR) ws expressed s (DWt DWo)/DWo where DWo is the dry weight just efore slt tretment nd DWt is the dry weight fter 15 dys of slt tretment. Dry weight (DW) ws mesured fter oven-drying smples t 7 for 48 h. Reltive wter ontent (RW) ws lulted s: RW (%) = (FW DW)/FW 1. Prior to determintion of fresh weight, shoots nd roots were wshed with distilled wter nd dried with towels. Free proline ontent ws mesured y the method of tes et l. (1973) using the ninhydrin regent. Proline onentrtion ws red t 52 nm nd determined from lirtion urve using L-Proline (Sigm ldrih) s stndrd nd expressed s nmol proline/g fresh weight Ion nlysis The N nd K, nd Mg ontents in the.rgusin roots nd shoots were determined y flme (PerkinElmer 6; Wlthm, M, US) nd grphite furne tomi sorption spetrophotometer (PerkinElmer 3) respetively, fter mirowve wet digestion (nton Pr Multiwve 3, Grz, ustri, EU) of the dried nd powdered mteril in 1 ml of supr-pure onentrted HNO 3 t 23. Estimtion ws rried out in triplite. l ws determined y the pplition of the oxygen flsk method followed y merurimetri titrtion with meruri perhlorte nd diphenilrzone s n inditor (Shöniger, 1955) Light mirosopy (LM) nd trnsmission eletron mirosopy (TEM) For ultrstruturl nlyses, smll piees of tissue were fixed for 3 min with 2% glutrldehyde in.5 M odylte uffer (ph 7.2) t 2. Upon rinsing with the odylte uffer, the mteril ws postfixed for 2 h with 1% (vol. rtio) osmium tetroxide in the sme uffer t 2. The mteril ws dehydrted through n ethnol series nd emedded in Spurr s resin. Semi-thin setions of fixed mteril were stined with 2% toluidine lue nd exmined using light mirosope Zeiss xiovert 35. Ultrthin setions were stined with urnyl ette nd led itrte nd exmined using FEI Morggni 268D eletron mirosope operted t n elerting voltge of 7 kv Mesurements of hlorophyll fluoresene, hlorophyll nd rotenoid ontent In vivo hlorophyll fluoresene ws mesured t room temperture with portle fluorometer (PM-2, Wlz, Germny) onneted to noteook omputer with dt quisition softwre (D-2, Heinz, Wlz). The plnt mteril ws drk-dpted for pproximtely 3 min efore mesurement. Estimtion ws rried out in triplite. The miniml (F o ) nd mximl fluoresene

3 S. Rdić et l. / Environmentl nd Experimentl otny 87 (213) levels were mesured in drk-dpted leves. The leves were then ontinuously illuminted with white tini light (photosyntheti photon flux density of 2 mol m 2 s 1 ) nd the sme prmeters were mesured (F nd F m). The rdition ws mintined until oth F nd F m were stle. lultions of fluoresene prmeters mximum quntum yield of the PS II (Fv/Fm), the effetive quntum yield of the PS II ( F/F m), nonphotohemil quenhing (NPQ) nd reltive eletron trnsport rte (reletr) were mde ording to Mxwell nd Johnson (2). Fresh mteril of dult leves ws extrted in 8% etone, nd ontents of hlorophyll (hl ), hlorophyll (hl ), nd rotenoids (r) were lulted ording to Lihtenthler (1987) Immunodetetion of Ruiso, D1, LHI, LHII nd FNR In order to determine the undne of proteins involved in photosynthesis, lef smples were homogenized in Tris Hl extrtion uffer ph 8 ontining 17.1% (w/v) surose,.1% (w/v) sori id,.1% (w/v) ysteine-hydrohloride (Sigm ldrih) with ddition of polyvinylpolypyrrolidone (PVPP, Sigm ldrih) nd then entrifuged t 25, g for 3 min. Totl protein onentrtion ws determined using ovine lumine serum s stndrd. liquots of eh homogente were mixed with orresponding volumes of denturting.65 M Tris Hl uffer ontining 6% (w/v) sodium dodeyl sulphte (SDS, Sigm ldrih), 6% (v/v) merptoethnol (Sigm ldrih), 3% (v/v) glyerol nd.1% (w/v) of romphenol lue, oiled for 5 min nd loded on the gel. The smples were seprted y SDS-polyrylmide gel eletrophoresis in 12% (w/v) resolving gels nd, susequently, eletrolotted onto the nitroellulose memrne (.45 m, io-rd). The memrnes were loked with 2.5% (w/v) non-ft powdered milk solution mde in Phosphte uffered sline (58 mm N 2 HPO 4, 17 mm NH 2 PO 4, 68 mm Nl) ph7.4 ontining 1% (v/v) of Tween 2 (Sigm-ldrih) nd inuted overnight with the following ntiodies rised ginst the pe proteins: rit nti-lrge suunit Ruiso (dilution 1:1); rit nti-d1 protein (dilution 1:25), rit nti-ferredoxin (Fd): NDPH oxidoredutse (FNR) (dilution 1:1), rit nti-light-hrvesting omplex II of PSII (LHII) (dilution 1:5) nd mouse nti-light-hrvesting omplex I of PSI (LHI) (dilution 1:5). Detetion of immunoretive proteins ws hieved y using lkline phosphtse-linked seondry ntiodies (dilution 1:3, nti-rit IgG from Sigm). The memrnes were developed with IP/NT (nitrolue tetrzolium nd 5-romo-4-hloro-3-indolyl phosphte (Sigm ldrih) ronyl groups nd sorte ontents The mount of protein oxidtion ws estimted y the retion of ronyl groups ( O) with 2, 4-dinitrophenylhydrzine (Sigm ldrih), s desried in Levine et l. (199). sorte ws estimted ording to the method of Mukherjee nd houdhouri (1983) using trihloroeti id nd dinitrophenyl hydrzyne nlysis of SOD, PX, T nd LOX tivities Shoot or root tissue ws homogenized in 5 mm KPO4 uffer (ph 7) inluding 5 mm sodium sorte, 1 mm ethylene dimine tetreti id (Sigm ldrih) nd PVPP. The homogentes were entrifuged (Sigm 3K18 entrifuge, Germny) t 25, g for 3 min t 4 nd superntnts used for enzyme tivity nd protein ontent ssys. Totl solule protein ontents of the enzyme extrts were estimted ording to rdford (1976) using ovine lumine serum (Sigm ldrih) s stndrd. The tivity of superoxide dismutse (SOD) ws ssyed y mesuring its ility to inhiit the photohemil redution of nitrolue tetrzolium (Sigm ldrih) following the method of euhmp nd Fridovih (1971). One unit of SOD ws tken s the volume of the enzyme extrt using 5% inhiition of nitrolue tetrzolium redution. sorte peroxidse (PX) tivity ws mesured ording to Nkno nd sd (1981). tlse (T) tivity ws determined y the deomposition of H 2 O 2 nd ws mesured following the method of ei (1984). Lipoxygense tivity ws determined using linoleni id s sustrte ording to xelrod et l. (1981) Sttistil nlysis For eh nlysis, dt were ompred y nlysis of vrine (NOV) with Dunn multiple Post Ho test using the STTISTI 1 (SttSoft, In., US) softwre pkge; differenes etween the orresponding ontrols nd exposure tretments were onsidered s sttistilly signifint t P <.5. Eh dt point is the verge of six replites (n = 6), unless stted otherwise. 3. Results 3.1. Reltive growth rte, reltive wter ontent nd proline ontent Mnnitol nd 15 mm Nl resulted in opposite effets on the growth of.rgusin plnts (Fig. 1) the first used growth inhiition (6 nd 2% derese of shoot nd root RGR, respetively, ompred to ontrol) nd the ltter used growth stimultion (lmost two-fold inrese of shoot nd root RGR ompred to ontrol). Plnt growth ws not ffeted y 3 mm Nl while higher Nl tretments, espeilly 6 mm, used signifint derese in the growth of.rgusin plnts (Fig. 1). Less onspiuous effets of slt were oserved on plnt reltive wter ontent (Fig. 1). Shoot RW of plnts exposed to 15 nd 3 mm Nl showed inrese y 3 nd 2%, respetively, while 6 mm Nl deresed shoot RW y 2% ompred to ontrol. derese of shoot RW ws highest following mnnitol tretment (13% ompred to ontrol). lthough root RW ws not ffeted y the lowest slt onentrtion, with the inrese of slt onentrtion, it grdully delined (3 6% in omprison to ontrol). Mnnitol-treted plnts lso showed 6% lower RW of roots. Regrding proline, the ontent of the mino id ws muh higher in roots thn in shoots of ontrol plnts. Slinity hd signifint effet on proline ontent in roots nd espeilly in shoots (Fig. 1). Proline ontent sustntilly inresed with n inrese in slinity, the inrese rnging from two-fold (t 15 mm) to 25-fold (t 6 mm) in shoots nd from 5% (t 15 mm) to three-fold (t 45 mm) in roots. Mnnitol used 2- nd three-fold inrese in shoot nd root proline ontent, respetively ontents of ions The ontent of hloride in ontrol plnts ws pproximtely ten times higher thn tht of sodium. Nevertheless, the ontents of oth ions, espeilly of N, inresed drmtilly with the mount of slt dded (Tle 1). Lef N ontent rehed mximum t 3 mm Nl it inresed up to 92 times ompred to tht of the ontrol while root N inresed signifintly with n inrese in slt onentrtion showing the highest vlue t 6 mm Nl (41-fold inrese ompred to ontrol). ontent of l in slt-treted plnts inresed mrkedly with n inrese in slt onentrtion; the lef l ws 6 to 1-fold higher nd tht of root 3 to 8-fold higher thn in respetive ontrols. The ontents of K, nd Mg in slt-treted plnts deresed ording to inresing slt onentrtions. The deline of those ions ws shrper in roots thn in shoots with the exeption of ; with inresed slinity K dropped for 3 5% in shoots nd 4 78% in roots, Mg dropped for 3 5% in shoots nd 4 67% in roots nd dropped for 35 65% in shoots nd 25 56%

4 42 S. Rdić et l. / Environmentl nd Experimentl otny 87 (213) () () () Reltive wter sttus(%) Reltive growth rte (g DW) Proline ( mol g -1 FW) e E e D shoot d root S1 S2 S3 S4 M Fig. 1. () RW (%), () RGR (g), nd () proline ontent of. rgusin shoots nd roots under ontrol () nd stress 15 mm Nl (S1), 3 mm Nl (S2), 45 mm Nl (S3), 6 mm Nl (S4), 3 mm mnnitol (M) onditions during the 15-dy growth peroid. Vlues re men ± SE sed on six replites. rs with different letters re signifintly different t p <.5. in roots. No signifint differenes in the ontents of N nd l were oserved etween ontrol nd mnnitol-treted plnts. The nonioni osmotium lso used derese of K, nd Mg whih ws more prominent in shoots. d D d 3.3. Lef morphology nd ntomy Exposure of. rgusin to Nl indued development of lef suulene (Fig. 2). Plisde nd spongy ells inflted under sline onditions, espeilly under 15 mm Nl, (Fig. 2) thus inresing the mesophyll thikness of. rgusin leves. The rise in lef thikness ws ompnied y deline in interellulr spes in mesophyll tissues (Fig. 2). ontrry to slt, mnnitol used prtil dethment of plsm memrne (Fig. 2, i) nd shrinkge of protoplsts inditing plsmolysis. The oservtion ws supported y the results of RW nd wilting of.rgusin leves (Fig. 2). The hloroplsts of the ontrol plnts were mostly ovlshped with few strh grins (Fig. 2d) nd their thylkoid memrnes were well developed (Fig. 2d). Under sline onditions, in ddition to normlly developed hloroplsts, the vesiulted hloroplsts with fewer numer of strh grins ut reltively still intt thylkoids were notied (Fig. 2e). Suh hloroplsts (Fig. 2e) were rrely oserved t 15 mm Nl while their numer inresed t 3 mm Nl. Under higher slt tretments (45 nd 6 mm Nl), thylkoid memrnes showed gret diltions nd undulted thylkoid res developed (Fig. 2f). The proportion of glyoxysomes (Fig. 2g) inresed with n inrese in slt onentrtion. lso, slinity indued vesiultion in the mesophyll ells (Fig. 2h). Vesiles were mostly fused with plsm memrne (Fig. 2f, h). Mnnitol-indued osmoti stress lso inresed the numer of memrne vesiles s well s of plstoglouls (Fig. 2i). However, the hloroplsts of mnnitol-treted plnts exhiited thylkoid memrnes with visile grn nd strom thylkoids Effiieny of PSII nd ontent of hlorophylls nd rotenoids The mximl effiieny of PSII (Fv/Fm) of slt-treted plnts deresed (y 14% ompred to ontrol) only in response to the highest slt onentrtion, while mnnitol suppressed Fv/Fm for 45% in omprison to ontrol (Fig. 3). Under sline onditions, the F/F m (Fig. 3) s well s ETR (Fig. 3d) dropped y similr mount with inresing slt onentrtion; the redution of the prmeters ws in the order 25 35% under lower Nl tretments, 6 7% under higher Nl tretments nd 8% under mnnitol (Fig. 3). ontrry to tht, nonphotohemil quenhing (NPQ) showed n inrese in the rnge of 46 78% under sline tretments (Fig. 3). There ws no signifint hnge in the NPQ of mnnitol-stressed leves (Fig. 3). Rising Nl slinity lso used ontinul derese in hlorophyll, hlorophyll, nd rotenoid ontent of.rgusin plnts (Fig. 3). When grown t 15 mm Nl, 35% nd 3% deline of hlorophylls nd rotenoids, respetively, in.rgusin plnts ws oserved (Fig. 3). t higher slinities, the redution of hl nd Tle 1 ontents of N, l, K, nd Mg (mg g 1 DW) of. rgusin plnts fter 15-dy period growth. Tretments N l K Mg Shoot ontrol 1.5 (.1) 18.8 (1.7)d 26.6 (2.8) 4. (.2) 1.2 (.2) 15 mm Nl 7.1 (8.6) (6.) 19.1 (3.5) 2.6 (.2).8 (.9) 3 mm Nl 138. (17.3) (5.9) 16.3 (2.9) 2.1 (.1).7 (.4) 45 mm Nl (11.5) (5.9) 13.7 (.8) 1.8 (.2).6 (.5) 6 mm Nl 12.4 (11.2) 198. (2.6) 12.3 (.9) 1.4 (.1).6 (.6) 3 mm mnnitol 1.2 (.1) 28.3 (5.)d 14.3 (.5) 1.4 (.2).6 (.6) Root ontrol 2. (.2)e 17.5 (2.6)d 3.3 (1.1) 1.6 (.3) 1.2 (.8) 15 mm Nl 22.7 (.8)d 55.2 (3.2) 17.9 (1.2) 1.2 (.3).7 (.2) 3 mm Nl 31.6 (2.1) 86.5 (6.9) 11.9 (.9)d 1. (.3)d.5 (.3)e 45 mm Nl 5.1 (3.1) 12.9 (13.8) 9.4 (.4)d.8 (.5)d.4 (.4)d 6 mm Nl 82.2 (8.6) (8.6) 6.8 (.4)e.8 (.4)d.4 (.3)d 3 mm mnnitol 2.2 (.1)e 23.5 (3.4)d 22.5 (.7) 1.5 (.8).8 (.5) Vlues represent men ± S.D. (prenthesis) of 3 replites. Different letters indite signifint differene t p <.1.

5 S. Rdić et l. / Environmentl nd Experimentl otny 87 (213) Fig. 2. Uppermost row morphology of. rgusin under ontrol () nd stress 15 mm Nl (S1), 3 mm Nl (S2), 45 mm Nl (S3), 6 mm Nl (S4), 3 mm mnnitol (M) onditions during the 15-dy growth period. LM photogrphs: ross-setions of leves () ontrol, () 15 mm Nl with strongly developed, inflted spongy prenhym, () mnnitol. TEM photogrphs: hloroplst of (d) ontrol, (e) 3 mm Nl-treted nd (f) 45 mm Nl-treted plnts; (g) glyoxysomes in Nl-treted ells, (h) vesiultion in Nl-treted ells, nd (i) mnnitol-treted ell. sp, spongy prenhym; v, vsulr undle; v, vuole; w, ell wll; pm, plsm memrne; vs, vesile; sg, strh grin; pl, plstogloul. r exeeded 5% in omprison to ontrol. Under sline onditions, degrdtion of hl ws fster (38% of the ontrol under 6 mm Nl) thn tht of hl nd r. Mnitol deresed hl, hl nd r y 35, 12 nd 22%, respetively, ompred to ontrol. Plnts grown t higher slinities, showed n inrese in hl / rtio whih ws signifint under 6 mm Nl (Fig. 3). Opposite to tht, mnnitol used 37% derese of the rtio. The hl + /r rtio ws not influened either y Nl or mnnitol (Fig. 3) Ruiso, D1, LHI, LHII nd FNR proteins Lrge suunit of Ruiso umulted less with inresing Nl onentrtions (Fig. 4). The intensity of other photosyntheti

6 44 S. Rdić et l. / Environmentl nd Experimentl otny 87 (213) Fig. 3. () hl, hl nd rotenoids ontent, () hl / rtio nd hl + /r rtio, () PS II effiieny Fv/Fm, F/F m nd NPQ (d) reletr of. rgusin leves under ontrol () nd stress 15 mm Nl (S1), 3 mm Nl (S2), 45 mm Nl (S3), 6 mm Nl (S4), 3 mm mnnitol (M) onditions during the 15-dy growth peroid. Vlues re men ± SE sed on six replites. rs with different letters re signifintly different t p <.5. protein nds strted to deline from 3 mm Nl with FNR showing the speifi degrding pttern. Mnnitol used lesser umultion of photosyntheti proteins ompred to ontrol while LHI protein ws not deteted under the tretment ronyl groups ontents nd LOX tivity The level of oxidtively dmged proteins, expressed s ronyl groups ontent, in.rgusin shoots inresed y 49 nd 47% under 45 nd 6 mm Nl, respetively, ompred to ontrol (Fig. 5). In roots, only the highest slt onentrtion used protein dmge (2-fold inrese of O ontent in omprison to ontrol). Mnnitol-treted plnts showed signifint inrese of O ontent in oth shoots nd roots (Fig. 5). The tivity of LOX in. rgusin shoots strted to inrese onsiderly from 45 mm Nl nd peked t 6 mm Nl while in roots only the highest slt onentrtion used signifint inrese of the enzyme (Fig. 5). Under mnnitol tretment, the inrese of LOX tivity in oth shoots nd roots exeeded 8% in omprison to ontrol (Fig. 5) ntioxidtive enzymes nd sorte ontent tivity of SOD in shoots ws signifintly inresed in response to lower Nl tretments (Fig. 5d). In roots, the tivity of SOD inresed with inresing slt onentrtions rehing the mximum t 6 mm Nl. The prodution of superoxide in mnnitol-treted plnts ws pproximtely 2-fold higher thn in ontrol plnts (Fig. 5d). Lower slt onentrtions signifintly inresed the tivity of T in oth shoots nd roots while higher ones did not ffet the tivity of the enzyme (Fig. 5e). Mnnitol used the indution of T in shoots while in roots the tivity ws similr to ontrol (Fig. 5e). The tivity of PX in shoots ws signifintly higher under 15, 3 nd 45 mm Nl thn in ontrol (Fig. 5f). In roots, higher slt onentrtions indued PX tivity ut only 45 mm Nl signifintly. Mnnitol inresed PX tivity in oth shoots nd roots y pproximtely 9% ompred to ontrol (Fig. 5f). The ontent of sorte in shoots ws signifintly lower (y pproximtely 4%) under 15, 3 nd 45 mm Nl thn in ontrol (Fig. 5). The ontent of sorte in roots ws ffeted only y the highest Nl onentrtion (27% lower vlue thn the ontrol). In shoots, mnnitol inresed sorte ontent y 43% while in roots the ontent of the ntioxidnt ws lowered y 25% in omprison to ontrol. 4. Disussion Fig. 4. Immunodetetion of Rl, FNR, LHI, LHII nd D1 proteins of. rgusin leves under ontrol () nd stress 15 mm Nl (S1), 3 mm Nl (S2), 45 mm Nl (S3), 6 mm Nl (S4), 3 mm mnnitol (M) onditions during the 15-dy growth peroid. Upon exposure to Nl,. rgusin umulted slt ions (Tle 1), inresed lef thikness nd produed suulent shoots regrdless of the slt onentrtion (Fig. 2). The inresed thikness of suulent leves n minly e ttriuted to enlrged mesophyll ells, whih hve sored wter long with slt ions nd inresed the size of their vuoles (Munns, 22). Rising the onentrtion of Nl in hydroponi solutions resulted in greter lef suulene nd greter mesophyll thikness for mny slt-tolernt plnts ut some non-hlophyti speies s well (Longstreth nd

7 S. Rdić et l. / Environmentl nd Experimentl otny 87 (213) () =O (nmol mg -1 protein) LOX (U mg -1 protein) () () SORTE ( mol g -1 FW) d shoot d root S1 S2 S3 S4 M SOD (U mg -1 protein) (d) T (U mg -1 protein) (e) PX (U mg -1 protein) (f) shoot root D d d S1 S2 S3 S4 M Fig. 5. () ronyl groups ontent ( O), () LOX tivity, () sorte ontent, (d) SOD tivity, (e) T tivity, nd (f) PX tivity of. rgusin shoots nd roots under ontrol () nd stress 15 mm Nl (S1), 3 mm Nl (S2), 45 mm Nl (S3), 6 mm Nl (S4), 3 mm mnnitol (M) onditions during the 15-dy growth peroid. Vlues re men ± SE sed on six replites. rs with different letters re signifintly different t p <.5. Noel, 1979; Mggio et l., 2; Silveir et l., 29). With respet to distriution of slt ions in different tissues, prevlent umultion of slt ions in leves versus roots suggests. rgusin to e n inluder (Yeo, 1983). However, regrding the type of inorgni ions preferentilly umulted,. rgusin employed N s primry osmolyte. Thus, in. rgusin osmoti djustment seems to e minly hieved y sodium nd, to lesser extent, hloride whih is onsistent with previous studies on slt tolernt speies (Wng et l., 1997; Khn et l., 2; Ued et l., 23; Silveir et l., 29). The umultion of slt ions expnded lef ells, positively ffeted plnt growth nd wter ontent ut only t 15 mm Nl (Fig. 1). threshold vlue for slt tolerne of the speies ws rehed t 3 mm Nl s ove tht onentrtion, inhiition of growth ws oserved. Similr growth pttern growth stimultion under reltively low slinity nd growth redution under high slinity ws oserved in mny slt tolernt nd hlophyti speies (jji et l., 1998; Short nd olmer, 1999; Koyro, 26). These results infer tht. rgusin ells seem to hve n effiient mehnism to djust osmotilly nd tht lef turgor is not limiting ftor for growth t moderte slinity levels. The reson for growth restrition t higher slinities might lie either in limited dilution pity of suulent tissue nd onsequent sturtion of the solute uptke system or in exessive demnd on the energy requirements of suh systems (Munns, 22). In ddition, the onomitnt inrese of proline with inresing slinity might hve ontriuted to negtive growth trend. It hs een shown tht metoli osts for osmoti djustment hieved y umultion of synthesized orgni solutes suh s proline re muh higher thn using Nl for the sme purpose (Munns, 22). The mssive proline umultion might hve ontriuted to mnnitol-indued growth inhiition of. rgusin s well. In ontrst to 15 mm Nl, iso-osmoti mnnitol restrited growth to the sme level s 45 mm Nl nd signifintly deresed lef wter ontent s well s lef thikness (Fig. 1). Thus, under osmoti stress used y mnnitol the inresing proline ontent did not result in n osmoti djustment level preventing plnt iomss redution nd wter loss. Similr effets of wter stress indued y either plnt non-irrigtion or y use of osmotium suh s mnnitol were oserved in other slt-tolernt speies s well (Ued et l., 23; Slm et l., 27). Derese of fresh weight nd wter ontent, s opposed to shrp inrese in proline ws lso reported under mnnitol-indued stress in hlophyte Sesuvium portulstrum (Slm et l., 27). Our results indite tht in.rgusin osmoti djustment through inorgni ion uptke is more effiient thn djustment through the prodution of proline. Severl uthors hve notied tht signifint proline umultion generlly ours only fter threshold of drought or slt stress is exeeded (vlieri nd Hung, 1979; Deluney nd Verm, 1993; Hester et l., 21). Therefore, in the se of. rgusin proline seem to serve other roles, suh s rdil svenging, protetion of ellulr mromoleules, storge of nitrogen or mintenne of ellulr ph (Verruggen nd Hermns, 28).

8 46 S. Rdić et l. / Environmentl nd Experimentl otny 87 (213) Other ftors, suh s nutrient defiienies, my lso ply n importnt role (Mrshner, 1995) in growth retrdtion. Disturne of nutrient lne is usul onsequene of either slinity or drought, irrespetive of plnt speies slt tolerne (Wng et l., 1997; Zekri, 1995; Ghoulm et l., 22; Slm et l., 27). Similr results were lso otined in our study s oth slt nd mnnitol deresed K, nd Mg ontents in.rgusin plnts. Drought nd slinity re found to distur the minerl-nutrient reltions in plnts through their effets on nutrient vilility, trnsport, nd prtitioning in plnts (Hu nd Shmidhlter, 25). In the present study, the effet of iso-osmoti Nl nd mnnitol on K, Mg nd ontents ws quite different with respet to plnt orgn - the nutrients in shoots were more ffeted y mnnitol nd those in roots were more ffeted y slt. Moreover, the mnnitol-indued imlne of lef nutrients ws omprle to tht used y higher slt onentrtions. Suh effets of higher slt tretments nd mnnitol might e ttriutle to redued nutrient uptke y the roots nd trnsport from the roots to shoots, respetively, s result of impired tive trnsport, redued ontent nd onsequent inresed memrne permeility (lm, 1999). In ddition, loss of turgor nd resultnt dehydrtion ould hve ontriuted to mnnitol-indued nutrient defiieny in. rgusin leves. Nevertheless, s oth drought nd slinity use similr effet on plnt growth through wter defiit, K is eqully importnt to mintin the turgor pressure of the plnt under either stresses. Derese of Mg uptke indued y slt nd mnnitol might hve ontriuted to deresed hlorophyll ontent nd thus impir photosyntheti mhinery (Hu nd Shmidhlter, 25). Severl studies hve suggested tht PSII is highly resistnt to slinity nd drought tress (Lu nd Zhng, 1998; orni nd Fresneu, 22; Deez et l., 28). In the present study, with the exeption of the highest slt onentrtion, sline onditions did not influene Fv/Fm rtios, mesured fter drk dpttion, inditing tht slinity does not indue sustined photodmge (Fig. 3). Unhnged Fv/Fm vlues were lso reported in kile mritim, Hordeum mritimum, triplex entrlsiti, Sorgum iolor (Qiu et l., 23; Netondo et l., 24; Megdihe et l., 28; Degl Innoenti et l., 29). However, the unimpired Fv/Fm vlues in slt-treted. rgusin, were ompnied y signifint derese in effetive quntum yield ( F/F m) nd redued eletron trnsport tivity. The redutions in F/F m nd reletr were orrelted with n inrese in NPQ whih my indite tht redued O 2 ssimiltion dereses demnd for produts of eletron trnsport, nd thus inreses therml dissiption of light energy t the ntenne. lthough, other mehnisms involved in energy dissiption relted to trnsmemrne proton grdient generted y TPse tivity nd unoupling of eletron trnsport tht my led to oxidtive stress, ould e lso involved (Mxwell nd Johnson, 2; ltyud nd rreno, 24). Struturl hnges of hloroplsts (Fig. 2) s well s deline of hlorophylls nd rotenoids (Fig. 3) oserved under sline onditions might hve ontriuted to impirment of retion enters of PSII either diretly (Msojidek nd Hll, 1992) or vi n elerted senesene (Kur-Hott et l., 1987). The hl / rtio nd the frequeny of vesiulted hloroplsts strted to inrese from 3 mm Nl nd ove tht onentrtion, slt used dmge to thylkoid memrnes nd deresed the grn stks, whih my finlly use the disturne or inhiition of photohemil retions. Moreover, these hloroplsts usully ontined little or no strh, suggesting low photosyntheti tivity (rhoumi et l., 27). n inrese in the hl / rtio under higher slinities might imply shift in the PSII/PSI rtio (Vrdi et l., 23) or rther derese in the LH omponents s hl is minly loted in the omplexes (Durnford et l., 23). This view is in greement with our results whih showed lesser undne of LHI, LH II nd D1 proteins strting from 3 mm Nl (Fig. 4). Slt stress ws found to inhiit the repir of the photodmged PSII through inhiition of the synthesis of proteins de novo nd, in prtiulr, the synthesis of the D1 protein (llkhverdiev et l., 22). Furthermore, deline of FNR nd Ruiso enzymes noted from 3 to 6 mm Nl indite lesser NDPH utiliztion nd photosyntheti ron redution whih might led to redued photosynthesis nd growth (Woodrow nd erry, 1988). ontrry to higher slt onentrtions, 15 mm Nl did not ffet the undne of immunodeteted proteins. lso, the ourrene of miroodies s well s of plstoglouls ws similr s in ells of ontrol plnts. Still, delined hlorophylls nd rotenoids nd the ourrene of modified hloroplsts might hve een the reson for downregultion of PSII. ompred to slt, mnnitol-indued osmoti shok used muh greter disturne to PSII; NPQ of mnnitol-treted leves showed no pprent hnge ompred to ontrol, though the vlue of the prmeter ws rther high in omprison to gretly deresed vlues of Fv/Fm, F/F m nd reletr. The downregultion of PSII ws prlleled with prtil or totl inhiition of LHs nd D1 proteins synthesis though the FNR nd Ruiso proteins umulted less s well (Fig. 4). In the study of Lu nd Zhng (1999) the loss of PSII hemistry under wter stress hs een ssoited with the loss or deline in D1 nd D2 proteins of PSII. The drsti effets of mnnitol on PSII nd some photosyntheti proteins might e sried to derese in RW whih hs een known to indue stomtl losure (orni nd Fresneu, 22; Reddy et l., 24). onsequent inhiition of O 2 ssimiltion, oupled with the hnges in photosystem tivities nd photosyntheti eletron trnsport pity, results in elerted prodution of tive oxygen vi the hloroplst Mehler retion (sd, 1999). oth slt nd mnnitol inresed the mount of memrne vesiles (Fig. 2) whih inrese the memrne surfe re nd re often disussed in onnetion with proesses suh s trnsport, storge, nd Nl omprtmenttion (Koyro, 22; Kurkov et l., 22; Mitsuy et l., 22). Higher slt onentrtions nd mnnitol ould ffet thylkoid memrnes y disrupting lipid ilyer or lipid protein ssoitions nd thus impir eletron trnsport tivity (Reddy nd Vor, 1986). Inresed numer of plstoglouls oserved under higher slt nd mnnitol tretments might e indition of stored lipid rekdown produts (Prmonov et l., 24; réhélin et l., 27). The formtion of plstoglouli is thought to e linked to the rekdown of thylkoids tht ompnies senesene (del Río et l., 1998). The umultion of miroodies suh s glyoxysomes, oserved under higher sline tretments is lso inditive of senesent proesses (Koyro, 1997). eside severl senesene-promoting ompounds suh s ethylene nd jsmoni id, importnt ftors in plnt senesene re retive oxygen speies nd LOX tivity whih, mong other, hs n importnt role in the rekdown of memrne lipids (del Río et l., 1998). Lipoxygense tlyzes the hydroperoxidtion of polyunsturted ftty ids with oxygen to give hydroperoxide produts whih n undergo utotlyti degrdtion, produing rdils nd thus inititing the hin retion of lipid peroxidtion. In ddition, LOX-medited formtion of singlet oxygen nd superoxide hs een shown (Knofsky nd xelrod, 1986; Lynh nd Thompson, 1984). Therefore, redued or unhnged LOX tivity under stress onditions n e onsidered s enefiil for plnts s LOX re oxidtive enzymes. In slt-tolernt tomto (Mittov et l., 22) or drought-tolernt hives (Egert nd Tevini, 22) deresed or redued LOX tivity hs een ompnied with unhnged mlondyldehide (inditor of lipid peroxidtion) ontent nd vie vers, inresed LOX tivity in drought-stressed olive (Sofo et l., 24) ws prlleled with inrese of MD ontent. Here, LOX tivity (Fig. 5) of oth shoots nd roots ws not ffeted y lower Nl tretments while it inresed in response to mnnitol nd higher slt onentrtions. However, lthough signifint inrese in the MD ontent ws oserved in. rgusin exposed

9 S. Rdić et l. / Environmentl nd Experimentl otny 87 (213) to either slt or mnnitol following 1 dys of stress, the level of lipid peroxidtion in either se ws unffeted fter 15-dy period (Rdić et l., 26). Some evidene suggests tht resistne to oxidtive stress hieved through effiient ntioxidnt defense mehnisms, my, t lest in prt, e involved in slt stress tolerne (Meloni et l., 23; en Hmed et l., 27; en mor et l., 25). In this study, indution of SOD, n enzyme whih elimintes superoxide nd simultneously produes H 2 O 2, ws reorded in oth plnt orgns under ll tretments exept in. rgusin leves exposed to higher slt onentrtions. The result implies tht oth slt or mnnitol inrese the formtion of superoxides in. rgusin leves nd roots proly s onsequene of the inhiition of lvin yle nd inresed respirtion in root mitohondri, respetively (Møller, 21; Mittov et l., 24). In plnts, T, PX nd GPX re onsidered the most importnt in degrdtion of H 2 O 2 (Prid nd Ds, 25). oth slt nd mnnitol indued the tivity of the enzymes, ut to different extent in plnt orgns. The enhnement in SOD, T nd POX tivities under slt nd wter stress ws lso reported in numer of hlophyti nd non-hlophyti speies (roetto et l., 22; Mittov et l., 22; Meloni et l., 23; Türkn et l., 25; en Hmed et l., 27). The oserved low levels of H 2 O 2 in slt-treted. rgusin leves (Rdić et l., 26) were mintined y PX nd T tivities whih were inresed only up to 3 mm Nl. The inresed PX tivity oinided with lowered sorte levels (Fig. 5) whih indite enzymti H 2 O 2 detoxifition i.e. sorte onsumption s n eletron donor for PX. In response to higher sline onditions, PX nd GPX (Rdić et l., 26) onstituted the system responsile for the elimintion of H 2 O 2, while T tivity ws inhiited, possily s result of ROS-indued degrdtion. Tht hypothesis ws orroorted with inresed level of oxidtively dmged proteins (Fig. 5) reorded in. rgusin leves under 45 nd 6 mm Nl. In roots, H 2 O 2 ws eliminted y T nd GPX under lower nd y PX under higher sline onditions, respetively. On the other hnd, the signifint inrese in H 2 O 2 (Rdić et l., 26) nd ronyl groups ontents (Fig. 5) ws reorded fter 15-dy period in response to mnnitol despite inresed GPX (Rdić et l., 26) nd PX tivities s well s high sorte ontent. Those results suggest tht.rgusin is equipped with lned nd responsive ntioxidnt system whih proved to e highly induile even under hyperosmoti onditions indued y mnnitol or hyperioni onditions rehed t higher slinities. In onlusion, the slt tolerne of this plnt speies n proly e ttriuted to its ility to: (1) develop lef suulene thus mintining onvenient tissue wter supply, (2) umulte nd omprtmentlize slt ions into the vuoles, (3) exhiit high ntioxidnt enzyme tivities preventing the toxi uildup of ROS. Nevertheless,. rgusin seems to e effiient t omprtmentlizing nd/or diluting slt ions only t sline levels not greter thn externl 3 mm Nl. The prtiulr threshold of. rgusin slinity tolerne proly lies in limited dilution pity of suulent tissue nd onsequent sturtion of the solute uptke system thus using oth hyperosmoti nd hyperioni stress, or in exessive demnd on the energy requirements of suh systems. knowledgments This study hs een funded y the rotin Ministry of Siene, Edution nd Sport, s prt of Projets no nd We grtefully knowledge the generous dontion of ntiodies y Dr. Hrvoje Fulgosi. Referenes ei, H., tlse in vitro. Methods in Enzymology 15, lm, S.M., Nutrient uptke y plnts under stress onditions. In: Pessrkli, M. (Ed.), Hndook of Plnt nd rop Stress. Mrel Dekker, New York, pp llkhverdiev, S.I., Nishiym, Y., Miyiri, S., Ymmoto, H., Ingki, N., Kneski, Y., Murt, N., 22. Slt stress inhiits the repir of photodmged photosystem II y suppressing the trnsription nd trnsltion of ps genes in Synehoystis. Plnt Physiology 13, rif, R., Küpeli, E., Ergun, F., 24. The iologil tivity of enture L. speies. G.U. Journl of Siene 17, sd, K., The wter wter yle in hloroplsts: svenging of tive oxygens nd dissiption of exess photons. nnul Review of Plnt Physiology nd Plnt Moleulr iology 5, xelrod,., heeserough, T.M., Lkso, S., Lipoxygense from soyens. E linolete:oxygen oxidoredutse. Methods in Enzymology 71, čić, T., Vidović, D., Popović, Ž., ontriution to knowledge out the lef ntomy of the rotin endemi txon enture rgusin L. susp. rgusin. Ntur roti 6, jji, M., Kinet, J.M., Lutts, S., Slt stress effets on roots nd leves of triplex hlimus L. nd their orresponding llus ultures. Plnt Siene 137, rhoumi, Z., Djeli, W., hïi, W., delly,., Smoui,., 27. Slt impt on photosynthesis nd lef ultrstruture of eluropus littorlis. Journl of Plnt Reserh 12, tes, L.S., Wldren, R.P., Tere, I.D., Rpid determintion of free proline for wter stress studies. Plnt nd Soil 39, euhmp,., Fridovih, I., Superoxide dismutse: improved ssy nd n ssy pplile to PGE. nlytil iohemistry 44, en mor, N., en Hmed, K., Deez,., Grignon,., delly,., 25. Physiologil nd ntioxidnt responses of the perennil hlophyte rithmum mritimum to slinity. Plnt Siene 168, , rtile/pii/s ff1. en Hmed, K., stgn,., Slem, E., Rnieri,., delly,., 27. Se fennel (rithmum mritimum L.) under slinity onditions: omprison of lef nd root ntioxidnt responses. Plnt Growth Regultion 53, or, M., Özdemir, F., Türkn, I., 23. The effet of slt stress on lipid peroxidtion nd ntioxidnts in leves of sugr eet et vulgris L. nd wild eet et mritim L. Plnt Siene 164, rdford, M.M., rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye inding. nlytil iohemistry 72, réhélin,., Kessler, F., vn Wijk, K.J., 27. Plstogloules: verstile lipoprotein prtiles in plstids. Trends in Plnt Siene 12, roetto, F., Lüttge, U., Rtjzk, R., 22. Influene of light intensity nd slttretment on mode of photosynthesis nd enzymes of the ntioxidtive response system of Mesemrynthemum rystllinum. Funtionl Plnt iology 29, ltyud,., rreno, E., 24. Response to ozone in two lettue vrieties on hlorophyll fluoresene, photosyntheti pigments nd lipid peroxidtion. Plnt Physiology nd iohemistry 42, vlieri,.j., Hung,.H.., Evlution of proline umultion in the dpttion of diverse speies of mrsh hlophytes to the sline environment. merin Journl of otny 66, orni, G., Fresneu,., 22. Photosyntheti ron redution nd ron oxidtion yles re the min eletron sinks for photosystem II tivity during mild drought. nnls of otny 89, Deez,., Koyro, H.W., Grignon,., delly,., Huzermeyer,., 28. Reltionship etween the photosyntheti tivity nd the performne of kile mritime fter long-term slt tretment. Physiologi Plntrum 133, Degl Innoenti, E., Hfsi,., Guidi, L., Nvri-Izzo, F., 29. The effet of slinity on photosyntheti tivity in potssium-defiient rley speies. Journl of Plnt Physiology 166, del Río, L.., Pstori, G.M., Plm, J.M., Sndlio, L.M., Sevill, F., orps, F.J., Jiménez,., López-Huerts, E., Hernndéz, J.., The tivted oxygen role of peroxisomes in senesene. Plnt Physiology 116, Deluney,.J., Verm, D.P.S., Proline iosynthesis nd osmoregultion in plnts. Plnt Journl 4, Durnford, D.G., Prie, J.., MKim, S.M., Srhfield, M.L., 23. Light-hrvesting omplex gene expression is ontrolled y oth trnsriptionl nd posttrnsriptionl mehnisms during photolimtion in hlmydomons reinhrdtii. Physiologi Plntrum 118, Egert, M., Tevini, M., 22. Influene of drought on some physiologil prmeters symptomti for oxidtive stress in leves of hives (llium shoenoprsum). Environmentl nd Experimentl otny 48, Flowers, T.J., Hjigheri, M.., lipson, N.J.W., Hlophytes. The Qurterly Review of iology 61, Ghoulm,., Foursy,., Fres, K., 22. Effets of slt stress on growth, inorgni ions nd proline umultion in reltion to osmoti djustment in five sugr eet ultivrs. Environmentl nd Experimentl otny 47, Hester, M.W., Mendelssohn, I.., MKee, K.L., 21. Speies nd popultion vrition to slinity stress in Pnium hemitomon. Sprtin ptens, nd Sprtin lterniflor: morphologil nd physiologil onstrints. Environmentl nd Experimentl otny 46, Hu, Y., Shmidhlter, U., 25. Drought nd slinity: omprison of their effets on minerl nutrition of plnts. Journl of Plnt Nutrition nd Soil Siene 168,

10 48 S. Rdić et l. / Environmentl nd Experimentl otny 87 (213) Knofsky, J.R., xelrod,., Singlet oxygen prodution y soyen lipoxygense isozymes. Journl of iologil hemistry 261, Khn, M.., Ungr, I.., Showlter,.M., 2. The effet of slinity on the growth, wter sttus, nd ion ontent of lef suulent perennil hlophyte, Sued frutios (L.) Forssk. Journl of rid Environment 45, Koyro, H.-W, Ultrstruturl nd physiologil hnges in root ells of Sorghum plnts (Sorghum iolor S. sudnensis v. Sweet Sioux) indued y Nl. Journl of Experimentl otny 48, Koyro, H.-W., 22. Ultrstruturl effets of slinity in higher plnts. In: Lühli,., Lüttge, U. (Eds.), Slinity: Environment Plnts Moleules. Kluwer demi Pulishers, Dordreht, The Netherlnds, pp Koyro, H.-W., 26. Effet of slinity on growth, photosynthesis, wter reltions nd solute omposition of the potentil sh rop hlophyte Plntgo oronopus (L.). Environmentl nd Experimentl otny 56, Kruse, G.H., Weis, E., hlorophyll fluoresene nd photosynthesis: the sis. nnul Review of Plnt Physiology nd Plnt Moleulr iology 42, Kur-Hott, M., Stoh, K., Ktoh, S., Reltionship etween photosynthesis nd hlorophyll ontent during lef senesene of rie seedlings. Plnt nd ell Physiology 28, Kurkov, E.., Klinkin, L.G., urin, O.K., Mysoedov, N.., Numov, T.G., 22. Responses of Seidlitzi rosmrinus to slt stress. iologil ulletin 29, Levine, R.L., Grlnd, D., Oliver,.N., mii,., liment, I., Lenz,.G., hn,.w., Shltiel, S., Stdtmn, E.R., 199. Determintion of ronyl ontent in oxidtively modified proteins. Methods in Enzymology 186, Lihtenthler, H.K., hlorophylls nd rotenoids: pigments of photosyntheti iomemrnes. Methods in Enzymology 148, Longstreth, D.V., Noel, P.S., Slinity effets on lef ntomy. Plnt Physiology 63, Lu,., Zhng, J., Thermostility of photosystem II is inresed in slt-stressed sorghum. ustrlin Journl of Plnt Physiology 25, Lu,., Zhng, J., Effets of wter stress on photosystem II photohemistry nd its thermostility in whet plnts. Journl of Experimentl otny 5, Lynh, D.V., Thompson, J.E., Lipoxygense-medited prodution of superoxide nion in senesing plnt tissue. FES Letters 173, Mggio,., Reddy, M.P., Joly, R.J., 2. Lef gs exhnge nd solute umultion in the hlophyte Slvdor persi grown t moderte slinity. Environmentl nd Experimentl otny 44, Mrshner, H., Minerl Nutrition of Higher Plnts, seond ed. demi Press, London. Msojidek, J., Hll, D.O., Slinity nd drought stress re mplified y high irrdine in sorghum. Photosyntheti 27, Mxwell, K., Johnson, G.N., 2. hlorophyll fluoresene prtil guide. Journl of Experimentl otny 51, Megdihe, W., Hessini, K., Ghri, F., Jleel,.., Ksouri, R., delly,., 28. Photosynthesis nd photosystem 2 effiieny of two slt-dpted hlophyti seshore kile mritim eotypes. Photosyntheti 46, Meloni, D.., Oliv, M.., Mrtinez,.., mri, J., 23. Photosynthesis nd tivity of superoxide dismutse, peroxidse nd glutthione redutse in otton under slt stress. Environmentl nd Experimentl otny 49, Mitsuy, S., Yno, K., Kwski, M., Tniguhi, M., Miyke, H., 22. Reltionship etween the distriution of N nd the dmges used y slinity in the leves of rie seedlings grown under sline ondition. Plnt Prodution Siene 5, Mittov, V., Tl, M., Volokit, M., Guy, M., 22. Slt stress indues up-regultion of n effiient hloroplst ntioxidnt system in the slt-tolernt wild tomto speies Lyopersion pennellii ut not in the ultivted speies. Physiologi Plntrum 115, Mittov, V., Guy, M., Tl, M., Volokit, M., 24. Slinity upregultes the ntioxidtive system in root mitohondri nd peroxisomes of the wild slt-tolernt tomto speies Lyopersion pennellii. Journl of Experimentl otny 55, Møller, I.M., 21. Plnt mitohondri nd oxidtive stress: eletron trnsport, NDPH turnover, nd metolism of retive oxygen speies. nnul Review of Plnt Physiology nd Plnt Moleulr iology 52, Mukherjee, S.P., houdhouri, M.., Implitions of wter stress-indued hnges in the levels of endogenous sori id nd hydrogen peroxide in Vign seedlings. Physiologi Plntrum 58, Munns, R., 22. omprtive physiology of slt nd wter stress. Plnt, ell & Environment 25, Murshige, T., Skoog, F., revised medium for rpid growth nd iossys with too tissue ultures. Plnt Physiology 15, N, O.., Tmme, M.., vri, M.., 28. Plynologil nd physiohemil hrteristis of three uniflorl honey types from entrl rgentin. Spnish Journl of griulturl Reserh 6, Nkno, Y., sd, K., Hydrogen peroxide is svenged y sorte-speifi peroxidse in spinh hloroplsts. Plnt nd ell Physiology 22, Netondo, G.W., Onyngo, J.., ek, E., 24. Sorghum nd slinity: II. Gs exhnge nd hlorophyll fluoresene of sorghum under slt stress. rop Siene 44, Prmonov, N.V., Shevykov, N.I., Kuznetsov, V.V., 24. Ultrstruture of hloroplsts nd their storge inlusions in the primry leves of Mesemrynthemum rystllinum ffeted y putresine nd Nl. Russin Journl of Plnt Physiology 51, Prid,.K., Ds,.., 25. Slt tolerne nd slinity effets on plnts: review. Eotoxiology nd Environmentl Sfety 6, Pieroni,., Jnik, V., Dürr,.M., Lüdeke, S., Trhsel, E., Heinrih, M., 22. In vitro ntioxidnt tivity of non-ultivted vegetles of ethni lnins in southern Itly. Phytotherpy Reserh 16, Qiu, N., Lu, Q., Lu,., 23. Photosynthesis, photosystem II effiieny nd the xnthophyll yle in the slt-dpted hlophyte triplex entrlsiti. New Phytologist 159, Rdić, S., Rdić-Stojković, M., Pevlek-Kozlin,., 26. Influene of Nl nd mnnitol on peroxidse tivity nd lipid peroxidtion in enture rgusin L. roots nd shoots. Journl of Plnt Physiology 163, Reddy, M.P., Vor,.., hnges in pigment omposition, Hill retion tivity nd shrides metolism in jr (Pennisetum typhoides S & H) leves under Nl slinity. Photosyntheti 2, Reddy,.R., hitny, K.V., Viveknndn, M., 24. Drought-indued responses of photosynthesis nd ntioxidnt metolism in higher plnts. Journl of Plnt Physiology 161, Rusk, G., Roinson, N., Pepeljnjk, S., 22. ntiteril nd ntifungl tivity of extrts nd queretgetin derivte isolted from enture rupestris L. (steree). t iologi roviensi Series otni 44, Shöniger, W., Eine mikronlytishe Shnellestimmung von Hlogen in orgnishen Sustnzen. Mirohimi t 43, Short, D.., olmer, T.D., Slt tolerne in the hlophyte Hlosri pergrnult susp. pergrnult. nnls of otny 83, Silveir, J..G., rújo, S..M., Lim, J.P.M.S., Viégs, R.., 29. Roots nd leves disply ontrsting osmoti djustment mehnisms in response to Nl-slinity in triplex nummulri. Environmentl nd Experimentl otny 66, 1 8. Slm, I., Ghny, T., Hessini, K., Messedi, D., Svoure,., delly,., 27. omprtive study of the effets of mnnitol nd PEG osmoti stress on growth nd solute umultion in Sesuvium portulstrum. Environmentl nd Experimentl otny 61, Slm, I., Ghny, T., Messedi, D., Hessini, K., Lidi, N., Svoure,., delly,., 27. Effet of sodium hloride on the response of the hlophyte speies Sesuvium portulstrum grown in mnnitol-indued wter stress. Journl of Plnt Reserh 12, Sofo,., Dihio,., Xiloynnis,., Msi,., 24. Lipoxygense tivity nd proline umultion in leves nd roots of olive trees in response to drought stress. Physiologi Plntrum 121, Türkn, I., or, M., Őzdemir, F., Ko, H., 25. Differentil response of lipid peroxidtion nd ntioxidnts in the leves of drought-tolernt P. utifolius Gry nd drought-sensitive P. vulgris L. sujeted to polyethylene glyol medited wter stress. Plnt Siene 168, Ued,., Knehi, M., Uno, Y., Ingki, N., 23. Photosyntheti limittions of hlophyte se ster (ster tripolium L.) under wter stress nd Nl stress. Journl of Plnt Reserh 116, Vrdi, G., Polynk, H., Drko, E., Lehozki, E., 23. trzine resistne entils limited xnthophylls yle tivity, lower PSII effiieny nd ltered pttern of exess exittion dissiption. Physiologi Plntrum 118, Verruggen, N., Hermns,., 28. Proline umultion in plnts: review. mino ids 35, Wng, L.-W., Showlter,.M., Ungr, I.., Effet of slinity on growth, ion ontent nd ell wll hemistry in triplex prostrt ouher. merin Journl of otny 84, Wng, D., Wng, H., Hn,., Wng,., Guo,., Zheng, D., hongjing, L., hng, L., Peng, M., Wng, X., 212. Sodium insted of potssium nd hloride is n importnt mronutrient to improve lef suulene nd shoot development for hlophyte Sesuvium portulstrum. Plnt Physiology nd iohemistry 51, Woodrow, I.E., erry, J.., Enzymti regultion of photosyntheti O 2 fixtion in 3 plnts. nnul Review of Plnt Physiology nd Plnt Moleulr iology 39, Zekri, M., PEG stress ltered itrus root nd lef minerl onentrtions. Journl of Plnt Nutrition 18, Yeo,.R., Slinity resistne: physiologies nd pries. Physiologi Plntrum 25,