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1 PERIODICUM BIOLOGORUM UDC 57:6 VOL., No, 5 58, CODEN PDBIAD ISSN -56 Originl sientifi pper Differentil esterse tivity in leves nd roots of Centure rgusin L. s onsequene of slinity SANDRA RADI] BRANKA PEVALEK-KOZLINA Deprtment of Botny Fulty of Siene University of Zgre Rooseveltov trg 6 Zgre, Croti Correspondene: Sndr Rdi} Deprtment of Botny Fulty of Siene University of Zgre Rooseveltov trg 6 Zgre, Croti E-mil: sndr@zg.iol.pmf.hr Key words: Centure rgusin, iso-esterse, slt stress, osmoti stress Astrt Bkground nd Purpose: Centure rgusin L. is n endemi Crotin plnt speies dpted to life in rid nd slt-ffeted environments. Esterse tivity (EST) nd isoesterse ptterns of C. rgusin plnts ultured in vitro under sline nd osmoti onditions were determined with n im to evlute the potentil vlue of esterse tivity s iomrker of slt/osmoti stress s well s physiologil signifine of EST vritions. Mteril nd Methods: Rooted plntlets grown on MS ½ nutrient medi supplemented with 5,, 45 or 6 mm NCl or mm mnnitol were investigted fter 5, nd 5 dys. Esterse tivity ws determined using either - or -nphthylette s sustrtes. Results nd Conlusion: Esterse tivities in shoots inresed under low sline nd mnnitol tretments nd deresed in response to inresed sline tretments. The highest slt nd onentrtion nd mnnitol stimulted EST tivity in roots. In totl, twelve nd fourteen esterse isoenzymes were resolved in C. rgusin leves nd roots, respetively. Both slt nd mnnitol indued new esterse isoenzyme (EST) while mnnitol speifilly indued two more (EST4-5); otherwise some nds were wekly expressed or even disppered s result of slinity in C. rgusin leves. Two new isosterses (EST-) were resolved in mnnitol- nd slt-treted roots nd four (EST, EST-4) only in slt-treted roots. Presented results demonstrte tht esterse tivities nd their isoenzymi ptterns ould serve s useful ioinditors of slinity. Reeived April 4, 8. INTRODUCTION All orgnisms tht exist within nturl environments re sujeted to stress t some point of their lives. The two mjor environmentl ftors tht urrently redue plnt produtivity re drought nd slinity whih oth use similr retions in plnts due to wter stress. The prolem is eoming more prevlent s the intensity of griulture inreses (). Therefore, it is importnt to understnd how plnts respond nd dpt to suh types of stress. Besides imposing osmoti stress, slinity indues ioni stress driven y toxi tion of N + nd Cl ions. Plnts need essentil minerl nutrients to grow nd develop. However, exessive solule slts in the soil re hrmful to most plnts. In ft, no toxi sustne restrits plnt growth more thn slt on world sle (). This hs led to reserh into slt tolerne with n im of improving rop plnts. Slt-tolernt plnts hve higher pity thn slt-sensitive ones to redue ytosoli N + y storing it in the vuole nd thus voiding sodium toxiity. Hyperosmolrity lso genertes se-

2 S. Rdi} nd B. Pevlek-Kozlin Effet of NCl nd mnnitol on C. rgusin esterse tivity ondry stress like oxidtive stress whih is used y exessive mounts of retive oxygen speies (ROS). ROS n hve dmging effet on ellulr strutures nd mromoleules lipids, proteins nd DNA. Besides nonenzymi ompounds of low moleulr weight, plnts hve developed numerous ntioxidtive enzymes ginst ROS (). Chnge (usully inrese) in the level of detoxifition enzyme tivity, suh s peroxidse, hs een used s potentil iomrker of mny toxi ompounds like hevy metls, pestiides, ut lso of stresses used y slinity, extreme tempertures, drought, ozone, et. (, 4, 5). In our previous study, peroxidse tivity of Centure rgusin L. ws mrkedly stimulted y mnnitol nd lso y NCl, though only up to mm (6). Two peroxidse isoenzymes were ommon to oth mnnitolnd slt-treted C. rgusin plnts while one isoenzyme ws indued y slt only. Here we investigted esterse tivity s potentil iomrker of slt stress using Centure rgusin L. s plnt model. As this plnt grow in the gpes of vertil limestone liffs long the ost nd on some islnds of Adriti Se, they re primrily ffeted y drought nd high light intensities nd indiretly y slinity vi sewter erosol (7). Esterses, group of hydrolses, tlyze the formtion or levge of ester onds of wter solule sustrtes. Generlly, these enzymes hve rod spetrum of sustrtes nd t on vriety of nturl nd xenoioti ompounds (8). Nphthylettes (- nd -), used for esterse tivity nd visuliztion in C. rgusin leves nd roots re sustrtes of rylesterses (EC...), whih re inhiited y sulfydryl regents nd prefer romti sustrtes, nd of roxylesterses (EC...) whih re inhiited y orgnophosphtes nd generlly prefer liphti esters (9). Esterses hve een extensively studied in insets nd vertertes ut muh less in plnts. Sine esterses exist in different isoenzymes in plnt nd niml tissues, their eletrophoreti pttern ws lso nlyzed. The reltionship etween esterse tivity nd slinity hs een investigted in severl plnt speies, irrespetive of their tolerne to slt (,, ). Thus, the im of the present study ws to estlish possile orreltion etween slt tolerne nd esterse tivity (EST) of C. rgusin plnts. Effets of isoosmoti onentrtions of NCl (5 mm) nd mnnitol ( mm) were ompred in order to disriminte possile differenes in C. rgusin response to ioni (NCl) nd non-ioni (mnnitol) omponent of slinity. MATERIALS AND METHODS C. rgusin seeds were olleted from nturl hitt on the islnd of Plgru`. The sterilized seeds were inoulted in test tues filled with 5 ml of MS ½ medium ontining. g L myo-inositol,. mg L thimine HCl,.5 mg L pyridoxine HCl,.5 mg L niotini id,.9 mm gierelli id (GA ),.5 mm 6-enzylminopurine (BA), g L surose nd 8 g L gr (). The shoots isolted from the seedlings were first suultured on the sme omposition medi. After 4 weeks in ulture, the shoots were trnsferred to liquid MS ½ medium ontining.5 mm indole--utyri id (IBA). Rooted plntlets were trnsferred to the sme omposition medi supplemented with different onentrtions of NCl (5 mm, mm, 45 mm or 6 mm) nd mnnitol ( mm), while ontrol plnts were kept in nutrient solution supplemented with IBA during the entire ssy. Enzyme extrtion nd solule protein determintion Lef (5 mg) nd root ( mg) smples were ground t 4 C using mortr nd pestle nd tissue extrts were prepred in n ie old 5 mm potssium phosphte (K HPO 4 /KH PO 4 ) uffer ph 7. ontining mm EDTA, with ddition of insolule polyvinylpyrrolidone (PVP-). The homogentes were entrifuged t g for min t 4 C (Sigm K8 Centrifuge). Superntnts were used for esterse tivity nd solule protein nlysis. Solule protein ontent ws determined y the method of Brdford using ovine serum lumin (BSA, Sigm) s stndrd (4). Esterse ssys Esterse tivity ws determined spetrophotometrilly t room temperture (5 C) using either - or -nphthylette s sustrtes (5). Esterse tivity ws lulted following the inrese in sorne t nm (for -nphthylette) or nm (for -nphthylette), due to the formtion of -nphtol (e nm =. mm m ) or -nphtol (e nm =.5 mm m ) (6). The retion mixture ontined. ml mm Tris/HCl ph 7.4 nd 8 ml mm - nphthylette or 5 ml mm - nphthylette, oth dissolved in solute methnol. For eh mesurement mlofrude extrt ws used. The enzyme tivity ws mesured every 5 seonds over three-min-period fter seond lg period. The esterse tivities were orreted for spontneous hydrolysis of - nd -nphthylette. The tivity ws expressed s mmol of hydrolysed sustrte per min per mg prot (mmol min mg protein ). Sttistil nlysis For eh nlysis, dt were ompred y nlysis of vrine (ANOVA), using STATISTICA 7. (SttSoft, In., USA) softwre pkge, nd differenes etween orresponding ontrols nd exposure tretment were onsidered s sttistilly signifint t P <.5. Eh dt point is the verge of nine replites. Ativity gel nlysis Tissue extrts were nlyzed eletrophoretilly under non-denturting onditions using vertil % polyrylmide sl gels with the uffer system of Lemmli without the SDS (7). A onstnt voltge of V ws pplied for 4 h nd the temperture mintined t 4 C. Equl mounts of protein (5 mg per well) were loded 54 Period iol, Vol, No,.

3 Effet of NCl nd mnnitol on C. rgusin esterse tivity S. Rdi} nd B. Pevlek-Kozlin onto eh lne. The esterse isozymes were visulized ording to modified proedure desried y Blen et l. (6). One- nd -nphthyl ettes, used s sustrtes (4 mg eh), were dissolved in 6 ml of 5% (v/v) etone nd mixed with ml of 5 mm Tris/HCl ph 7.. After stining ( min), the gels were wshed with tp wter nd inuted in solution ontining 5 mm Tris/HCl ph 7. nd Fst Blue RR slt until drk rown (-nphthyl ette) or purple red (-nphthyl ette) nds ppered ( min). The Fst Blue RR slt ( mg) ws dissolved in ml of solute methnol nd filtered into 5 mm Tris/HCl ph 7.. The gels were one more rinsed with tp wter nd fixed in % (v/v) ethnol. RESULTS Morphology of C. rgusin plnts Morphologil pperne of C. rgusin leves grown in the presene of isoosmoti onentrtions of slt (5 mm) or mnnitol ( mm) nd without either of them (ontrol) in vitro onditions is presented in Figure. Compred to ontrol plnts, C. rgusin stems nd leves eome fleshy nd suulent under sline onditions. Following hyperosmoti stress indued y mnnitol, plntlets lost freshness nd turgesene, grdully wilted nd eme yellow. Esterse tivity of C. rgusin plnts Esterse tivity ws evluted with two sustrtes, - nd -nphthylette. Generlly, esterse tivity of C. rgusin leves mesured with -nphthylette (-EST) deresed with inresing slt onentrtions (Figure ), i.e. -EST tivity signifintly inresed in response to the lowest slt tretment (5 mm NCl), showed no hnge in response to mm NCl, nd deresed in response to higher slt tretments (45 nd 6 mm). Mnnitol used onsiderle inrese in -EST tivity in C. rgusin leves fter nd 5 dys. In roots, -EST tivity ws silly ffeted only y the highest slt tretment fter nd 5 dys nd y mnnitol fter 5 dys; otherwise the tivity ws similr to ontrol. The esterse tivity of C. rgusin leves ssyed with -nphthylette (-EST) ws lmost two times lower ompred to the tivity ssyed with -nphthylette (Figure ). After nd 5 dys, lower (5 nd mm) nd higher (45 nd 6 mm) slt onentrtions used inrese nd derese in lef -EST tivity, respetively. In roots, -EST tivity ws fter 5 dys elevted in response to mm NCl. A signifint inrese in -EST tivity of C. rgusin roots ws otined t 6mM NCl fter nd 5 dys. Osmoti stress used y mnnitol ontinuously inresed -EST tivity of oth leves nd roots. Esterse isoenzymes of C. rgusin plnts Results presented in Figure show vritions in the esterse isoenyzme pttern of C. rgusin leves nd roots under mnnitol nd different slt tretments. In totl, twelve esterse isoenzymes were resloved in C. rgusin leves d d d d d d Esterse tivity (-nphthylette) roots C S S S S4 M C S S S S4 M C S S S S4 M 5. dy. dy 5. dy Figure. Lef morphology of tissue ultured ontrol, slt- (5 mmncl) nd mnnitol-ffeted C. rgusin plnts. Figure. Esterse tivity (mmol min mg protein ) during 5-dy growth period with -nphthylette s sustrte in C. rgusin leves nd roots under ontrol (C) nd stress 5 mm NCl (S), mm NCl (S), 45 mm NCl (S), 6 mm NCl (S4), mm mnnitol (M) onditions. Vlues re men ± S.E. sed on six replites. Brs with different letters re signifintly different t P <.5. Period iol, Vol, No,. 55

4 S. Rdi} nd B. Pevlek-Kozlin Effet of NCl nd mnnitol on C. rgusin esterse tivity leves Esterse tivity (-nphtylette) roots C S S S S4 M C S S S S4 M C S S S S4 M 5. dy. dy 5. dy Figure. Esterse tivity (mmol min mg protein ) during 5-dy growth period with nphthylette s sustrte in C. rgusin leves nd roots under ontrol (C) nd stress 5mM NCl (S), mm NCl (S), 45 mm NCl (S), 6 mm NCl (S4), mm mnnitol (M) onditions. Vlues re men ± S.E. sed on six replites. Brs with different letters re signifintly different t P <.5. leves ut only four (EST, EST6-8) were ommon to ll extrts. Those isoenzymes umulted less under high slt tretments. Isoesterses EST nd EST were present fter five dys in ll lef extrts nd fter nd 5 dys in ontrol nd 5 mm NCl-treted leves. Mnnitol indued EST4 nd EST5 fter nd 5 dys. Isoesterse EST9 ws oserved s fint nd in ll lef extrts fter 5 dys nd ws sent in ontrol nd mnnitol-treted leves fter nd 5 dys. Isoesterse EST ws expressed in ontrol nd slt-treted leves nd ws sent under mnnitol trtment. Its olortion ws intensive in response to ontrol fter 5 dys nd in response to lower slt onentrtions during ll the time. Isoesterse EST ws indued only fter 5 dys y higher slt onentrtions nd mnnitol. Isoesterse EST ws notied in ll extrts fter 5 nd dys. After 5 dys it ws present only under higher slt nd mnnitol onentrtions. Five lef EST isozymes (EST, EST6-9) reted with -nphthyl ette whih gve drk rown olortion, two (EST, EST) reted with -nphthyl ette whih gve purple red olortion nd four isoesterses (EST, EST4-5, EST) reted with oth sustrtes (dt not shown). Although fourteen esterse isoenzymes were resloved in C. rgusin roots, only six (EST, EST-4, EST6-8) were oserved in ll extrts during the entire oservtion period. Isoesterses EST nd EST4 umulted Figure 4. Eletrophoreti ptterns of lef nd root esterse isoenzymes under ontrol nd stress onditions during 5-dy growth period. Gels were inuted in solution of oth sustrtes together. Equl mount of proteins (5 mg per well) ws loded onto eh gel lne. C-ontrol, S-5 mm NCl, S- mm NCl, S-45 mm NCl, S4-6 mm NCl, M- mm mnnitol. more in response to mnnitol. Isoesterse EST5 ws visile fter 5 nd dys in ll extrts wheres fter 5 dys it ppered only in response to 6 mm NCl. Slt indued isoenzymes EST9 nd EST. Isoenzyme EST9 ppered s fint nd fter 5 dys under lower slt onentrtions nd fter nd 5 dys t 6 mm. Isoenzyme EST ws notied s fint nd under ll slt tretments exept the highest one under whih it ws more intensely expressed. Both slt nd mnnitol indued isoenzymes EST nd EST t 5 dys ut the isoenzymes were more pronouned under mnnitol stress. Root isoesterses EST, EST nd EST4 were speifilly indued y 6 mm NCl fter 5 dys of growth. Under tht tretment, very intensive olortion of isoenzymes EST5-9 ws notied. Almost hlf of root EST nds were drk rown (EST, EST5-9) nd the other hlf purple red (EST-4, EST-, EST4) depending on the retion with -nphthyl ette nd -nphthyl ette, respetively. Three isoesterses (EST, EST-) reted with oth sustrtes (dt not shown). DISCUSSION The response of plnts to slt stress is sed on the tion of mny defense proteins/enzymes (8). Plnt isoesterses nd esterse tivity hve een relted to hevy 56 Period iol, Vol, No,.

5 Effet of NCl nd mnnitol on C. rgusin esterse tivity S. Rdi} nd B. Pevlek-Kozlin metl nd pestiide toxiity, pthogenesis, morphogenesis nd emryogeni potentil (9,, ). In the present study, esterse tivity nd isoesterse pttern were studied in leves nd roots of C. rgusin sujeted to NCl- nd mnnitol-indued stress. In this plnt speies, esterse tivity differed less with regrd to sustrtes thn to plnt orgns, s with oth - nd -nphthylette the tivity in leves ws muh more ffeted thn tht in roots. Stimultion of lef esterse tivity ws evident under lower (5 nd mm NCl) slt onentrtions (Figure, ). Similrly, mung en (Vign rdit) nd Sued mritim plnts, ultured in in vitro onditions, exhiited the highest esterse tivity etween 5 nd 4 mm NCl (). Inrese of esterse tivity ws lso oserved in Lemn minor plnts (dukweed) exposed to led, dmium, hromium, zin, opper nd merury (). In the study, uthors imply esterse vrition s well s the indution of severl new esterse isoenzymes to e dptive mehnism of dukweed to metl ontmintion. In greement with tht view, stimultion of C. rgusin esterse tivity with inresing slt tretments ould e relted to slt tolerne of the speies hieved through ion umultion (6). However, s higher slt onentrtions (45 nd 6 mm NCl) suppressed oth -nd -EST of C. rgusin leves, positive orreltion etween esterse tivity nd slt onentrtion seems to exist only up to s high s mm NCl. It hs een demonstrted tht slt onentrtions higher thn 4 mm NCl inhiit most enyzmes euse of the perturtion of the hydrophoi-eletrostti lne etween the fores mintining protein struture (8). Opposite to tht, esterse tivities of slt-treted roots showed lmost no differene in omprison to ontrol prt from the highest slt (6 mm NCl) onentrtion whih used onsiderle inrese in - nd -EST tivities. Comprison of isoosmoti NCl (5 mm) nd mnnitol ( mm) onentrtions, it is evident tht mnnitol used muh greter stimultion of esterse tivity in oth C. rgusin leves nd roots. The result might reflet different osmoregultion of C. rgusin to osmoti stress used y NCl nd mnnitol. Suulent nd enlrged leves nd stems of C. rgusin oserved on sline medi, suggests tht C. rgusin uses slt ions s reltively heper omptile solute in omprison to orgni solutes suh s proline or surose (6). Thus, y muh greter esterse tivity deteted in C. rgusin plnts exposed to mnnitol-indued osmoti stress, the ells would e provided with enough omponents nd/or energy needed for synthesis of orgni solutes. Indeed, the metoli osts i.e the numer of moles of ATP for osmoti djustment hieved y umultion of synthesized orgni solutes, proved to e muh higher thn using NCl insted (). Mnnitol- -indued growth inhiition of C. rgusin plnts notied in our previous study (4) might e explined y higher prodution of orgni solutes. The esterse pttern in C. rgusin displyed tissue speifiity. The numer of isoenzymes in roots ws higher thn in leves of ontrol plnts. Another exmple of the vriility oserved etween lef nd root esterses is their differene in sustrte preferene. Esterse tivity mesured with -nphthyl ette (-EST) in C. rgusin leves ws two times higher thn the tivity mesured with -nphthyl ette (-EST). This orresponded to isoesterse pttern of C. rgusin leves where the mjority of nds were drk rown nd only few were purple red fter gel olortion with -nphthyl ette nd/or -nphthyl ette, respetively. Oppositely, preferene of esterse towrd prtiulr sustrte ws not oserved in C. rgusin roots. Vriility etween lef nd root esterses ws lso oserved under slt stress. Generlly, the numer of isoesterses in C. rgusin leves ws the sme s in ontrol while tht of roots inresed under the influene of NCl nd mnnitol. Slt indued one new isoenzyme in leves nd seven in roots of C. rgusin plnts. The esterse ptterns of penut (Arhis hypoge L.) roots exposed to slinity were lso more ffeted ompred to those in leves (). In the study, slt stress indued 4 5 isoenzymes in penut roots versus three new isoenzymes in its leves fter two-week period. However, some of slt-indued esterse isoenzymes in C. rgusin roots (EST nd ) were lso indued y mnnitol whih points to some differenes in C. rgusin response to ioni nd osmoti omponent of slinity. In ddition, two esterse isoenzymes (EST4, EST5) in C. rgusin leves resulted only from mnnitol-tretment. Mjority of new isoesterses ppered s lte s t the end of the two-week period (only few ppered fter dys), suggesting de novo synthesis of new esterse isoenzymes. In onfirmtion with the postulte, indution of esterse tivity used y mnnitol nd slt ws notied fter nd espeilly fter 5 dys. However, some sline tretments lso indued new isoenzymes lthough esterse tivities were deresed or similr to ontrol. The wilting of C. rgusin plnts oserved upon mnnitol tretment nd development of suulene in response to isoosmoti onentrtion of slt implies tolerne of C. rgusin to NCl-indued osmoti stress. In onlusion, isoesterses proved to e more suitle iomrkers of slt/osmoti stress thn isoperoxidses s greter numer of speifilly indued isoenzymes ws oserved in C. rgusin esterse pttern (ten in totl) thn in its peroxidse pttern (six in totl). Thus, the presented results demonstrte tht esterse tivities nd their isoenzymi ptterns ould serve s useful ioinditors of slinity. Aknowledgements: This study ws supported y Crotin Ministry of Siene, Edution nd Sport, Projet no REFERENCES. BOHNERT H J, NELSON D E, JENSEN R G 995 Adpttions to environmentl stresses. Plnt Cell 7: 99. BERNSTEIN N, SILK W K, LÄUCHLY A 995 Growth nd development of sorghum leves under onditions of NCl stress: possi- Period iol, Vol, No,. 57

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