Salinity and drought represent serious problems worldwide negatively

Transcription

1 PERIODICUM BIOLOGORUM UDC 57:61 VOL. 11, No 3, 93 99, 1 CODEN PDBIAD ISSN Originl sientifi pper Effets of osmoti stress on ntioxidtive system of dukweed (Lemn minor L) SANDRA RADI] BRANKA PEVALEK-KOZLINA Deprtment of Botny Fulty of Siene University of Zgre Rooseveltov trg 6 1 Zgre, Croti Correspondene: Sndr Rdi} Deprtment of Botny Fulty of Siene University of Zgre Rooseveltov trg 6 1 Zgre, Croti E-mil: sndr@zg.iol.pmf.hr Key words: oxidtive dmge, PEG, mnnitol, NCl, ntioxidnt, proline Arevitions: APOX sorte peroxidse; CAT tlse; POX pyrogllol peroxidse; MDA mlondildehyde; PEG polyethylene glyol Astrt Bkground nd Purpose: It is known tht osmoti stress my use dmge to ells y induing tive oxygen speies prodution or y disrupting detoxifition mehnisms. We hypothesize tht inresed tivity of ntioxidnt enzymes in dukweed (Lemn minor L.) provides mehnism of tolerne to osmoti stress. Mteril nd Methods: Plnts were sujeted to NCl- (5 mm), mnnitol- (5 nd 1 mm) nd polyethylene glyol-medited osmoti stress (PEG, 5 mm) for period of 15 dys. The responses of ntioxidtive system inluding lso hnges in growth, proline ontent nd the extent of oxidtive dmge in terms of mlondildehyde, H O nd hlorophyll to hlorophyll (hl /) nd hlorophyll + to rotenoids (hl +/r) rtios were studied. Results nd Conlusion: Iso-osmolr onentrtions of slt nd mnnitol signifintly redued reltive growth rte ompred to ontrol plnts while osmoti shok medited y PEG used omplete esstion of growth. Proline ontent inresed with the severity of osmoti stress showing the highest vlues in PEG-treted plnts. The proline umultion upon PEG stress ws prlleled y equl inrese of sori id pool. Ctlse, sorte peroxidse nd non-speifi peroxidse tivities showed onsiderle inrese under ll osmoti gents, espeilly PEG. The inresed enzyme tivities oinided with unhnged H O levels, hl / nd hl +/r under mnnitol nd slt stress. Despite the highest indution of ntioxidtive defense, mrked inrese in lipid peroxidtion nd H O level s well s derese of hl / nd hl +/r ompnied PEG tretment. These results suggested tht indution of ntioxidnt defenes is t lest one omponent of the tolerne mehnism of plnts to long-term osmoti stress. Reeived April 16, 8. INTRODUCTION Slinity nd drought represent serious prolems worldwide negtively influening plnt growth in griulturl nd nturl eosystems. Aording to Munns (1) growth is limited predominntly y osmoti effet, ommon oth to slinity nd drought, while slt-speifi effets develop with time. Depending on the speies, plnts del with suh effets in very verstile mnners inluding intrellulr omprtmenttion of slt nd osmoti djustment hieved y umultion of inorgni ions or orgni solutes suh s proline (). However, osmoti stress might lso use stomtl losure nd redue CO fixtion led-

2 S. Rdi} nd B. Pevlek-Kozlin Effets of osmoti stress on dukweed (Lemn minor L) ing to shortge of NADP + whih serves s n eletron eptor in photosynthesis. Consequent leking of eletrons from eletron trnsport hins to moleulr oxygen enles inresed prodution of retive oxygen speies (ROS) suh s singlet oxygen, superoxide, hydrogen peroxide nd hydroxyl rdils whih n e lso formed in mitohondri, peroxisomes nd plsm memrnes (3, 4). These ytotoxi ROS re highly retive nd n distur norml metoli proesses through oxidtive dmge of lipids, proteins nd nulei ids. As ounter-t, plnts hve evolved effiient defense mehnisms onsisting of low moleulr mss ntioxidnts sori id, rotenoids, proline, toopherols nd ROS-interting enzymes superoxide dismutse (EC ), tlse (CAT, EC ), nd vriety of peroxidses (sorte peroxidse APOX, EC ; non-speifi peroxidse POX, EC ). Aside from ting s n osmolyte nd n ntioxidnt, mny other funtions hve een ssigned to proline suh s stiliztion of proteins nd regultion of the ytosoli ph vlue (5). Mny studies show positive orreltion etween enhned ntioxidtive system nd inresed resistne to osmoti stress nd mny other environmentl stresses (6, 7, 8). Dukweed (Lemn minor L.) is smll floting mrophyte ommon to wide rnge of rivers nd lkes ll over the world (9). Mny dvntges suh s rpid growth, genetilly homogenous popultions, esiness of ulture nd sensitivity to mny toxi sustnes mke dukweed suitle plnt model. The reltive degree of L. minor tolerne to osmoti stress ws ssessed y studying the hnges in ntioxidnt system nd hl / nd hl +/ r rtios (1). Severl osmoti gents differing y their physio-hemil properties hve een used to simulte effets of osmoti stress in vitro PEG (non-ioni-impermele), mnnitol (non-ioni-permele) nd NCl (ioni-permele). As PEG simultes wter defiit onditions in ultured ells in similr mnner to tht oserved in the ells of intt plnts sujeted to true drought onditions, the use of PEG of high moleulr weight (6 or 8) is reommended (11). A few studies rried out on Lemn plnts orrelted tivtion of ntioxidnt system to oxidtive injury used y short- -termed NCl stress (1), s well s redution of growth to proline umultion in response to different osmoti gents (13). However, involvement of ntioxidtive defense system in mehnisms of oxidtive dmge y PEG- nd mnnitol-indued osmoti stress hs not yet een studied in L. minor plnts. We hypothesized tht dukweed tolerne to osmoti stress ould e ssoited with the mintenne of or indution of ntioxidtive system. Hene, this study reports 1) omprtive nlysis of the hnges in the non-enzymti (sori id nd rotenoids) nd enzymti (tivities of CAT, APOX nd POX nd their eletrophoreti ptterns) ntioxidtive omponents in test plnts upon PEG nd iso-osmolr onentrtions of mnnitol nd NCl, nd ) possile indution of oxidtive stress upon PEG nd mnnitol. MATERIALS AND METHODS Plnt mteril nd ulture Lemn minor L. ws originlly olleted from Botnil Grden, Fulty of Siene, University of Zgre. Plnts were sterilized ording to Krjn~i~ nd Devidé (14) nd mintined s stok ultures on Pirson-Seidel nutrient medium (15). The ph vlue of nutrient medi ws djusted to 4.55 with.1 M KOH nd medi utolved t 118 kp nd 1 C for mins. The ultures were grown under 16 hour photoperiod of fluoresent light (8 mem - s -1 )t4± C. Growth prmeters Individul helthy olonies with -3 fronds (from stok ultures) were trnsferred to 1 m 3 Erlenmyer flsks ontining 6 m 3 Pirson-Seidel (PS) nutrient medium eh supplemented with 5 mm NCl, 5 mm mnnitol (M 5 ), 1 mm mnnitol (M 1 )or5mm PEG (MW 6) exept in the se of ontrol. The ulture flsks were mnully shken one dy. Frond numers were monitored during 15-dy period, on dys, 3, 5, 8, 1,1 nd 15 (n). Reltive frond numer (RFN) ws lulted using the eqution (16): RFN = (frond No. t dy n frond No. t dy )/ frond No. t dy. Reltive growth rte (RGR) ws sed on fresh mss (f.m.) determined fter 15 dys of experiment nd lultedusingthefollowingformul: RGR=[ln (finl weight) ln (initil weight)]/ weeks. Chlorophyll nd rotenoid ontent Photosyntheti pigments were mesured ording to the method desried y Arnon (17). In short, 3 mg fresh smples were homogenized with 8% (w/v) old etone, entrifuged t 5 g for 1 min nd sornes of the superntnt t 663, 646 nd 47 nm were red. Chlorophyll, nd totl rotenoid ontent ws determined ording to Lihtenthler (18). MDA nd H O ontent Lipid peroxidtion ws determined y estimting the mount of mlondildehyde (MDA) ontent using the thiorituri id method desried y Heth nd Pker (19). The rude extrts were mixed with.5% (w/v) thiorituri id solution ontining 1% (w/v) trihloroeti id, heted t 95 C for 3 min nd the retion ws stopped in n ie th. The ooled mixtures were entrifuged t 1 g for 1 min nd the MDA ontent lulted from the sorne t 53 nm (orretion ws done y sustrting the sorne t 6 nm for non-speifi turidity) y using extintion oeffiient of 155 mm m.h O ws extrted from 1 mg fresh tissue in ie-old etone nd estimted ording to the method of Mukherjee nd Choudhuri (). After ddition of titnyl-sulphte nd on. NH 4 OH solution, the formed peroxide-titnium preipitte ws dissolved inmh SO 4 nd sorne of the mixtures red t 415 nm. The H O ontent ws lulted from stndrd urve nd expressed s mmol (H O )g (f.m.). 94 Period iol, Vol 11, No 3, 1.

3 Effets of osmoti stress on dukweed (Lemn minor L) S. Rdi} nd B. Pevlek-Kozlin Asorte ontent sorte ws isolted y extrtion with 6% trihloroeti id from 1 mg plnt tissue, following the method of Mukherjee nd Choudhuri (). Extrts were mixed with % dinitrophenyl hydrzyne followed y the ddition of 1 drop of 1% thioure solution nd oiled for 15 min in wter th. After dding 8% (w/v) H SO 4 (in n ie-th), the sorne of the mixtures ontining hydrzone omplex ws red t 53 nm. Asorte onentrtion ws determined using lirtion urve nd expressed s mmol (sorte) g (f.m.). Proline ontent Free proline ontent ws mesured y the method of Btes et l. (1). Plnt tissue (1 mg) ws homogenized in 3% (w/v) sulphoslyyli id nd entrifuged t 7 g for 3 min. After ddition of ninhydrin regent, mixtures were heted t 1 C for 1 h nd ooled in n ie-th. The hromophore otined ws extrted from liquid phse with toluene nd the sorne of orgni lyer ws red t 5 nm. Proline onentrtion ws determined from lirtion urve using L-Proline s stndrd nd expressed s nmol (proline) g (f.m.). Enzyme determintions For enzyme nlysis, ultures were strted y trnsferring 5 helthy olonies with 3 fronds from stok ultures in 5 m 3 Erlenmyer flsks ontining 1 m 3 PS medium supplemented with NCl, mnnitol nd PEG-6 in the sme onentrtions s in growth experiments. Plnt tissue (1 mg) ws homogenized in 1m 3 1 mm potssium phosphte (KPO 4 ) uffer (ph 7) inluding 1 mm EDTA nd polyvinylpolypyrrolidone (PVPP), using pre-hilled mortrs nd pestles. The homogentes were entrifuged t g formint 4 C. The superntnt ws used for enzyme tivity nd protein ontent ssys. Totl solule protein ontents of enzyme extrts were estimted ording to Brdford () using ovine lumine serum (BSA, Sigm) s stndrd. Asorte peroxidse tivity ws determined ording to Nkno nd Asd (3). The sorte oxidtion ws followed t 9 nm nd its onentrtion lulted using the molr extintion oeffiient (e =.8 mm m ). Corretions were done for low, non-enzymti oxidtion of sorte y H O. One enzyme unit ws defined s mmol (oxidized sorte) g (f.m.) per min. Ctlse (CAT) tivity ws determined y the deomposition of H O nd ws mesured spetrophotometrilly y following the derese in sorne t 4 nm (4). Ativity ws lulted using the extintion oeffiient (e =.4 mm m ) nd mmol (H O deomposed) g (f.m.) min ws defined s unit of CAT. The tivity of non-speifi peroxidse ws mesured y monitoring the formtion of purpurogllin t 43 nm (e =.47 mm m ) ording to Chne nd Mehly (5). The retion mixture ontined 5 mm KPO 4 uffer (ph 7), 1mM H O, mm pyrogllol nd enzyme extrt. Speifi enzyme tivity for ll enzymes ws expressed s units mg protein. Isoenzyme profile of APOX, POX nd CAT Smples of L. minor extrts were seprted y non- -denturting gel eletrophoresis in 1% (APOX) or 7% (CAT) polyrylmide sl gels t ph 8.3 nd 4 C ording to Lemlli (6). Equl mounts of protein (5 mg for APOX, 4 mg for CAT, 3 mg for POX) were loded onto eh lne. For detetion of APOX isoforms, the gels were first soked in 5 mm KPO 4 uffer (ph 7) ontining mm sorte for 3 min (3 x min). The gels were then inuted in the sme uffer inluding 4 mm sorte nd mm H O for min, riefly wshed nd the hromti nds visulized in 5 mm KPO 4 uffer (ph 7.8) ontining.45 mm NBT nd 8 mm TEMED. Following 3 min inution in 5 mm KPO 4 uffer (ph 7.), visuliztion of POX tivity ws performed in the sme uffer ontining mm pyrogllolnd4mmh O (7). Stining for CAT ws rried out s desried y Woodury et l. (8). After 3 wshes for 15 min in distilled wter, the gels were soked in 5 mm H O for 1 min. After rief rinse, the gels were inuted in 1% (w/v) ferri hloride / 1% potssium ferriynide solution nd CAT isozymes ppered s olorless nds on drk green field. Sttistil nlysis Dt were nlyzed y one-wy nlysis of vrine (ANOVA) using Sttisti 7.1. (SttSoft, In.) 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 (n=9). MDA [ nmol g (f.m.) ] C C C C S M5 M1 P Figure 1. H O (line) nd MDA (olumn) ontents in L. minor plnts under ontrol (C) nd stress 5mM NCl (S), 5 mm (M 5 ) or 1 mm mnnitol (M 1 ) nd 5mM PEG onditions fter 15-dy growth period. Vlues re men ± S.E. sed on nine replites. Brs with different letters re signifintly different t p <.5. B A H O [ mol g (f.m.) ] Period iol, Vol 11, No 3, 1. 95

4 S. Rdi} nd B. Pevlek-Kozlin Effets of osmoti stress on dukweed (Lemn minor L) ASCORBATE [m mol g (f m) ] PROLINE [ nmol g (f m) ] d d C S M5 M1 P Figure. Asori id () nd free proline () ontents under ontrol (C) nd stress 5 mm NCl (S), 5 mm (M 5 ) or 1 mm mnnitol (M 1 ) nd 5mM PEG onditions fter 15-dy growth period. Vlues re men ± S.E. sed on nine replites. Brs with different letters re signifintly different t p <.5. RESULTS Effets of NCl, mnnitol nd PEG on L. minor growth, in terms of reltive frond numer nd reltive growth rte re shown in Tle 1. Applition of 5 mm mnnitol used reltive frond numer retrdtion from dy 1, ut hd no effet on reltive growth rte. Iso- -osmolr onentrtions of NCl, nd mnnitol signifintly redued reltive growth rtes nd reltive frond numers (from dy 8), oth to lmost the sme extent. PEG used totl growth inhiition. Chlorophylls nd rotenoid ontents displyed the highest vlues in response to mnnitol, no signifint hnge upon NCl nd mrked derese upon PEG tretments (Tle ). Ch / nd hl +/r rtios deresed for 18% nd 8%, respetively, in L. minor plnts exposed to PEG. The hnges of MDA nd H O ontents under tested osmoti gents re given in Figure 1. The plnts treted with 5 mm NCl nd mnnitol exhiited no inrese in H O nd MDA levels, while 1 mm mnnitol tretment inresed MDA formtion y nerly 7% ompred to ontrol, though it did not hnge H O ontent. Mximum MDA (15%) nd H O (33%) vlues, s ompred to ontrol, were oserved under PEG stress, inditing high extent of lipid peroxidtion. Asori id nd proline ontents re shown in Figure. Asori id pool mrkedly (71% ompred to ontrol) inresed under PEG tretment while other osmoti gents exhiited vlues similr to ontrol (Figure A). Iso-osmolr NCl nd mnnitol tretments signifintly inresed proline ontent in L. minor plnts, ut not to suh high level s PEG (16% when ompred to ontrol) (Figure B). Figures 3A-C desrie the effet of osmoti stress on APOX, POX nd CAT tivities in L. minor plnts fter 15 dys of exposure to tested osmoti gents. POX tivity of plnts exposed to NCl ws similr to ontrol ut inresed y 31% nd 7% under 5 nd 1 mm mnnitol, respetively, nd 87% under PEG tretment (Figure 3A). All osmoti gents signifintly inresed APOX tivity in tested plnts NCl nd higher onentrtion of mnnitol y 45%, 5 mm mnnitol y 3%, nd PEG y 16% (Figure 3B). The indution of CAT ws most onspiuous under PEG nd 5 mm mnnitol tretments pproximtely 4% inrese ompred to ontrol (Figure 3C). Higher onentrtion of mnnitol nd NCl inresed the tivity of the enzyme y pproximtely 3% nd %, respetively. The POX, APOX nd CAT isoenyzme ptterns of L. minor plnts sujeted to different osmoti tretments re presented in Figure 4. In totl, four POX isoenzymes ould e diserned on ntive gels (Figure 4A). Isoenzyme POX4 ws not present in ontrol ut ppered upon stress imposition. The intensity of isoenzyme POX4 inresed in the order NCl < M 5 <M 1 < PEG. TABLE 1. Growth of L. minor plnts grown in PS medium without (ontrol) or with 5 mm NCl, 5 mm mnnitol (M5), 1 mm mnnitol (M1) nd 7 gl -1 PEG for period of 15 dys. Reltive growth rte (fter 15 dys) Reltive frond numer 3rd dy 5th dy 8th dy 1th dy 1th dy 15th dy Control.189±.14.±.5 1.± 3.19± ± ± ±1.88 NCl.16±.11.17±.1.86±.31.38± ± ± ±.4 M5.18±.8.4±.47 1.±.15.88± ± ± ±.11 M1.16±.9.5±.4.94±.75±.7 5.± ± ±1.33 PEG Vlues re mens of six replites ± S.D. per tretment. Mens in eh olumns followed y different letters re signifintly different (p<.5). 96 Period iol, Vol 11, No 3, 1.

5 Effets of osmoti stress on dukweed (Lemn minor L) S. Rdi} nd B. Pevlek-Kozlin 5 POX [ Um g (protein) ] APOX [ Um g (protein) ] 3 1 CAT [ Um g (protein) ] C S M5 M1 P Figure 3. Non-speifi peroxidse (), sorte peroxidse () nd tlse () tivities under ontrol nd stress onditions fter 15-dy growth period. Vlues re men ± S.E. sed on nine replites. Brs with different letters re signifintly different t p <.5. For revitions see Figure. The intensity of two oserved APOX isozymes inresed ording to the hnge in enzyme tivity, eing the most intense upon PEG-indued stress (Figure 4B). Out of three oserved tlse isozymi nds, CAT ws missing in ontrol nd CAT3 umultion inresed under tretments (Figure 4 C). Figure 4. Eletrophoreti ptterns of non-speifi peroxidse (), sorte peroxidse () nd tlse () isoenzymes under ontrol nd stress onditions during 15-dy growth period. For revitions see Figure. DISCUSSION In the present study, osmoti gents PEG, NCl nd mnnitol indued differentil ntioxidnt nd growth responsesin model plnt systeml. minor. Long-term exposure (15 dys) to mild osmoti tretment used inhiition of lonl reprodution (presented s reltive frond numer) y nerly %, ut hd no effet on reltive growth rte (sed on fresh mss). The pplition of iso-osmolr NCl nd mnnitol onentrtions reveled no signifint differene etween the effets of these two osmoti gents on growth in oth ses reltive frond numers nd reltive growth rtes were ffeted y pproximtely 5% nd 15%, respetively (Tle 1). In ddition, the most negtive impt on L. minor growth indued y non-ioni-impermele PEG suggests it is the osmoti nd not ioni omponent of slinity tht is responsile for onsidering dukweed slt-sensitive plnt TABLE. Content of hlorophyll, nd rotenoids [mg/g (f.m.)], hlorophyll + rtio nd hlorophyll +/rotenoids rtio in L. minor plnts sujeted to different osmoti for 15-dy period. For revitions see Tle 1. Tested medi hl hl rotenoids hl / hl +/rotenoids Control.58±..±.1.7±.1.91±.15.88±.16 NCl.56±.7.18±..7±.1.8±.8 3.±.1 M5.64±.3.±..3±.1.96±.3.91±.16 M1.65±..3±.1.31±..88±.16.89±.7 PEG.47±.5.19±..4±.3.4±.1.66±.8 Vlues re mens of six replites ± S.D. per tretment. Mens in eh olumn followed y different letters re signifintly different (p<.5). Period iol, Vol 11, No 3, 1. 97

6 S. Rdi} nd B. Pevlek-Kozlin Effets of osmoti stress on dukweed (Lemn minor L) speies (9). Similr NCl/mnnitol nd PEG effet on L. minor growth were otined y Frik nd Golt (13) who lso notied orreltion etween deresed growth rte nd inresed proline umultion. Our dt onfirmed these results sine the highest proline ontent with mximum growth inhiition ws oserved under PEG nd the lowest with miniml effet on growth under 5 mm mnnitol (Figure B). It hs lredy een shown tht metoli osts for osmoti djustment hieved y umultion of synthesized orgni solutes suh s proline re muh higher thn using NCl for the sme purpose nd thus tend to led to growth inhiition (3, 31). Besides their primry effets on growth nd photosynthesis, the negtive effets of slt nd wter stresses my e in prt onsequene of oxidtive dmge to importnt moleules, resulting from the imlne etween ROS formtion nd ntioxidnt defenses. In this study, ll osmoti gents inresed APOX, CAT nd POX tivities in L. minor plnts, ut the indution of studied enzymes ws most onspiuous under PEG-indued stress (Figure 3A-C). As onfirmtion, new CAT isoform nd inresed umultion of APOX nd CAT isoforms were reveled y ntive PAGE under stress onditions (Figure 4). Inrese of APOX, CAT nd POX, s mjor H O -detoxifying enzymes, hs een relted to etter slt- nd wter defiit-resistne in mny plnt speies (8, 3). Compring tivities of studied enzymes in osmotilly stressed L. minor plnts, POX nd espeilly APOX tivities were greter thn CAT tivity. This my e so euse 1) peroxidses hve muh higher ffinity for H O thn tlse, nd ) sorte-peroxidse is regrded s one of the most widely distriuted ntioxidnt enzymes in plnt ells, eing present in the ytosol, hloroplsts, miroodies, mitohondri, s well s in the ell wll (33). MDA, seondry end produt of free rdil indued lipid peroxidtion nd H O ontents re tken s mesures of the stress-indued dmge t ellulr level (34). H O is nturl plnt metolite tht exerts its detrimentl role predominntly y genertion of highly retive hydroxyl rdils whih then initite lipid peroxidtion. On the other hnd, H O ts s seond messenger to regulte the gene expression of some ntioxidtive enzymes in plnt ells, nd trnsient inrese of H O is oserved during the erly stges of oxidtive stress (35). PndndUpdhyy (1) reportedonsiderle H O umultion in dukweeds exposed to short- -term slinity, whih ws ompnied y derese in CAT tivity. Thus, in the present study, H O might hve up-regulted ntioxidtive system of L. minor plnts under NCl- nd mnnitol- indued stresses sine fter -week-period CAT, APOX nd POX tivities were enhned to suh n extent tht they in turn ensured unhnged H O levels (Figure 1). It hs lso een shown tht sorte nd rotenoids re le to intert diretly with ROS (superoxide, hydroxyl rdils nd singlet oxygen), preventing them from inititing lipid peroxidtion (36). Thus, together with the ove mentioned results, elevted nd/or unffeted rotenoid nd sorte ontents s well s unhnged rtios of hl / nd hl +/r spek ginst oxidtive dmge under moderte osmoti stress, even though the MDA ontent inresed y 7% in response to 1 mm mnnitol. However, it is evident tht in spite of remrkly high sorte nd ntioxidnt tivity, PEG tretment eqully inresed MDA nd H O levels nd deresed hl nd rotenoid ontents, suggesting tht the extent of oxidtive injury is muh higher thn the dukweed defense mehnisms. Also, the rtios of hl / nd hl +/r deresed under PEG-medited stress, whih implies slower degrdtion of hl. Chl plys n importnt role in stilizing poproteins nd the ssemly of light hrvesting omplex (37). Severl studies hve shown tht PEG promotes plnt senesene (38, 39). Generlly, hl rekdown is ommon event during senesene nd it is ssumed tht hl loss is linked to protein degrdtion (4). An inresed rte of senesene nd tivity of different proteolyti enzymes ws notied in whet nd rie exposed to osmoti stress (41). In our study, PEG my hve promoted premture senesene lso euse tht osmoti gent used not only the degrdtion of hl ut lso of protein ontent in dukweed plnts (dt not shown). It is noteworthy tht mssive proline umultion oserved under PEG-indued stress ould not meliorte onsequent oxidtive dmge nd inhiitory effets on L. minor growth, though it might hve ontriuted to more effiient ntioxidnt enzyme tivity (4). Shrm nd Duey (33) found tht ddition of externl proline prevents APOX intivtion under PEG-indued wter defiit nd suggested the protetive role of proline for enzyme tivity under osmoti stress onditions. Effets of PEG s n osmoti gent were fr more severe thn those of mnnitol during 15 dys of stress imposition, proly euse of slight sorption of mnnitol. While PEG nnot enter the pores of plnt ells, mnnitol hs een shown to e tken up y plnt ells where it n serve s omptile solute (43, 44). This ould e the reson for the sene of, or low extent of, lipid peroxidtion under mnnitol-indued osmoti stress. In onlusion, our hypothesis ws onfirmed y the results of this study whih showed tht tolerne of L. minor to long-term moderte osmoti stress ws losely relted to stimultion of ntioxidnt defense mehnisms. The results lso show dukweed pity to ope with severe osmoti stress though t the expense of omplete growth esstion. Aknowledgements: This study ws supported y Crotin Ministry of Siene, Edution nd Sport, s prt of Projet no REFERENCES 1. MUNNS R Comprtive physiology of slt nd wter stress. Plnt Cell Environ 5: AJMAL KHAN M, UNGAR I A, SHOWALTER A M Effets of slinity on growth, wter reltions nd ion umultion of the 98 Period iol, Vol 11, No 3, 1.

7 Effets of osmoti stress on dukweed (Lemn minor L) S. Rdi} nd B. Pevlek-Kozlin sutropil perennil hlophyte, Atriplex griffithii vr. Stoksii. Ann Bot 85: HALLIWELL B 1987 Oxidtive dmge, lipid peroxidtion nd ntioxidnt protetion in hloroplsts. Chem Phys Lipids 44: HERNANDEZ J A, CORPAS F J, GOMEZ M, DEL RIO L A, SEVILLA F 1993 Slt-indued oxidtive stress medited y tivted oxygen speies in pe lef mitohondri. Physiol Plnt 89: HONG Z, LAKKINENI K, ZHANG Z, VERMA DP Removl of feedk inhiition of delt (1) pyrroline-5-roxylte synthetse results in inresed proline umultion nd protetion of plnts from osmoti stress. Plnt Physiol 1: LOPEZ F, VANSUYT G, CASSE-DELBART F, FOURCROY P 1996 Asorte peroxidse tivity, not the mrna level, is enhned in slt-stressed Rphnus stiv plnts. Physiol Plnt 97: MITTLER R, ZILINSKAS B A 1994 Regultion of pe ytosoli sorte peroxidse nd other ntioxidnt enzymes during the progression of drought stress nd following reovery from drought. Plnt J 5: TÜRKAN I, BOR M, ÕZDEMIR F, KOCA H 5 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 Si 168: LEWIS M A 1995 Use of freshwter plnts for phytotoxiity testing: A review. Environ Pollut 87: SHAW B P 1995 Chnges in the levels of photosyntheti pigments in Phseolus ureus Rox. exposed to Hg nd Cd t two stges of development: omprtive study. Bull Environ Contm Toxiol 55: AHMADMSA,JAVEDF,ASHRAF M 7 Iso-osmoti effet of NCl nd PEG on growth, tions nd free proline umultion in llus tissue of two indi rie (Oryz stiv L.) genotypes. Plnt Growth Regul 53: PANDA S, UPADHYAY R K 4 Slt stress injury indues oxidtive lterntions nd ntioxidtive defense in the roots of Lemn minor. Biol Plnt 48: FRICK H, GOLT C 1995 Sensitivity of Lemn minor growth to osmoti potentil nd reltive tolerne of its llus. J Plnt Physiol 146: KRAJN^I^ B, DEVIDÉ Z 198 Report on photoperiodi responses in Lemnee from Sloveni. Ber Geoot Inst 47: PIRSON A, SEIDEL F 195 Cellulr nd physiologil investigtions of Lemn minor root with speil onsidertion to potssium nd lium lk. Plnt 38: ENSLEY H E, BARBER J T, POLITO M A, OLIVER A I 1994 Toxiity nd metolism of,4-dihlorophenol y the quti ngiosperm Lemn gi. Environ Toxiol Chem 13: ARNON D I 1949 Copper enzymes in isolted hloroplsts: Polyphenoloxidse in Bet vulgris. Plnt Physiol 4: LICHTENTHALER H K 1987 Chlorophylls nd rotenoids: Pigments of photosyntheti memrnes. Methods Enzymol 148: HEATH RL, PACKER L 1968 Photoperoxidtion in isolted hloroplsts. I-kinetis nd stoihiometry of ftty id peroxidtion. Arh Biohem Biophys 15: MUKHERJEE S P, CHOUDHURI M A 1983 Implitions of wter stress-indued hnges in the levels of endogenous sori id nd hydrogen peroxide in Vign seedlings. Physiol Plnt 58: BATES L S, WALDREN R P, TEARE I D 1973 Rpid determintion of free proline for wter stress studies. Plnt Soil 39: 5 7. BRADFORD M M 1976 A rpid nd sensitive method for the quntittion of mirogrm quntities of protein utilizing the priniple of protein-dye inding. Anl Biohem 7: NAKANO Y, ASADA K 1981 Hydrogen peroxide is svenged y sorte-speifi peroxidse in spinh hloroplsts. Plnt Cell Physiol : AEBI H 1984 Ctlse in vitro. Methods Enzymol 15: CHANCE B, MAEHLY A C 1955 Assy of tlses nd peroxidses. In: Colowik S P, Kpln N O (eds) Methods in Enzymology. Ademi Press, New York, p LAEMLLI U K 197 Clevge of struturl proteins during the ssemly of the hed of teriophge T4. Nture 7: MITTLER R, ZILINSKAS B 1993 Detetion of Asorte Peroxidse Ativity in Ntive Gels y Inhiition of the Asorte-Dependent Redution of Nitrolue Tetrzolium. Anl Biohem 1: WOODBURY W, SPENCER A K, STAHMANN M A 1971 An improved proedure using ferryynide for deteting tlse isozymes. Anl Biohem 44: HILLMAN W S 1961 The Lemnee or dukweeds. Bot Rev 7: PÉREZ-ALFOCEA F, ESTAÑ M T, CARO M, GUERRIER G 1993 Osmoti djustment in Lyopersion esulentum nd L. pennellii under NCl nd polyethylene glyol 6 iso-osmoti stresses. Physiol Plnt 87: RAVEN J A 1985 Regultion of ph nd genertion of osmolrity in vsulr plnts: ost-enefit nlysis in reltion to effiieny of use of energy, nitrogen nd wter. New Phytol 11: ROUT N P, SHAW B P 1 Slt tolerne in quti mrophytes: possile involvement of the ntioxidtive enzymes. Plnt Si 16: SHARMA P, DUBEY RS 4 Asorte peroxidse from rie seedlings: properties of enzyme isoforms, effets of stresses nd protetive roles of osmolytes. Plnt Si 167: LIN C C, KAO C H Effet of NCl stress on H O metolism in rie leves. Plnt Growth Regul 3: FOYER C H, LOPEZ-DELGADO H, DAT J F, SCOTT I M 1997 Hydrogen peroxide- nd glutthione-ssoited mehnisms of limtory stress tolerne nd signlling. Physiol Plnt 1: NOCTOR G, FOYER C H 1998 Asorte nd glutthione: keeping tive oxygen under ontrol. Ann Rev Plnt Physiol & Plnt Mol Biol 49: PLUMLEY F G, SCHMIDT G W 1995 Light-hrvesting hlorophyll / omplexes: Interdependent pigment synthesis nd protein ssemly. Plnt Cell 7: BEHERA S K, NAYAK L, BISWAL B 3 Senesing leves possess potentil for stress dpttion: the developing leves limted to high light exhiit inresed tolerne to osmoti stress during senesene. J Plnt Physiol 16: PANDEY R, AGARWAL R M, JEEVARATNAM K, SHARMA G L 4 Osmoti stress-indued ltertions in rie (Oryz stiv L.) nd reovery on stress relese. Plnt Growth Regul 4: HASHIMOTO H, KURA-HOTTA M, KATOH S 1989 Chnges in protein ontent nd in the struture nd numer of hloroplsts during lef senesene in rie seedlings. Plnt Cell Physiol 3: SRIVALLI B, KHANNA-CHOPRA R 1998 Drought indued enhnement of protese tivity during monorpi senesene in whet. Curr Si 75: JAIN M, MATHUR G, KOUL S, SARIN NB 1 Ameliortive effets of proline on slt stress indued lipid peroxidtion in ell lines of groundnut (Arhis hypoge L.). Plnt Cell Rep : ABEBE T, GUENZI A C, MARTIN B, CUSHMAN JC 3 Tolerne of mnnitol-umulting trnsgeni whet to wter stress nd slinity. Plnt Physiol 131: STOOP J M H, WILLIAMSON J D, PHARR D M 1996 Mnnitol metolism in plnts: method for oping with stress. Trends Plnt Si 1: Period iol, Vol 11, No 3, 1. 99