Effect of Salt Stress on Growth, Membrane Damage, Antioxidant Metabolism and Artemisinin Accumulation in Artemisia annua L.

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1 Plnt Strss 21 Glol Sin Books Efft of Slt Strss on Growth, Mmrn Dmg, Antioxint Mtolism n Artmisinin Aumultion in Artmisi nnu L. Triq Aft * M. Msroor A. Khn Moh. Irs M. Nm Nm Hshmi Moinuin Plnt Physiology Stion, Dprtmnt of Botny, Aligrh Muslim Univrsity, Aligrh , Ini Corrsponing uthor: * trik.lig@gmil.om ABSTRACT Slinity in soil n irrigtion wtr is on of th mjor ftors tht limit rop proutivity. Th ffts of slinity on growth, lipi proxition, th ntioxint fn systm n hngs in rtmisinin ontnt wr stui in Artmisi nnu L. Slinity trtmnts wr stlish y ing,, 1, n 2 mm of soium hlori (NCl) to th soil. Slt strss ngtivly fft th growth of plnts, msur in trms of shoot n root lngth n ry wight. Photosynthti ttriuts n totl hlorophyll ontnt wr lso ru y slinity strss. Slinity trtmnts inhiit th tivity of roni nhyrs n signifintly inrs ltrolyt lkg n prolin ontnt. Morovr, slt strss inu oxitiv strss, s init y th lvl of lipi proxition. Th tivitis of ntioxint nzyms viz. tls (CAT), proxis (POX) n suproxi ismuts (SOD) wr uprgult y slt strss. Most importntly, synrgisti rltionship ws not twn nognous H 2 O 2 n rtmisinin ontnt i.. oth ontnts inrs unr low lvls of slinity ( n 1 mm) n thrftr rs. Thus, it n onlu tht A. nnu spis is vry snsitiv to soil slinity; howvr, mort slin onitions n utiliz to otin mor rtmisinin. Kywors: ltrolyt lkg, lipi proxition, photosynthsis, slinity strss INTRODUCTION Agriulturl proutivity is svrly fft y soil slinity n th mging fft of slt umultion in griulturl soils hs om n importnt nvironmntl onrn. Evry yr mor n mor ln oms non-proutiv u to slt umultion. In Ini, of th 9.38 million h of slt-fft soil, 3.88 million h r lkli soil n. million h (inluing ostl lns) r slin soil (Jll t l. 27). Plnts grown in griulturl systms r xpos to mny nvironmntl strsss limiting thir yil potntil. Slinity is on of th limiting nvironmntl ftors for soil frtility n plnt proution. Exss slt in soil my vrsly fft plnt growth ithr through osmoti inhiition of wtr uptk y roots or spifi ion ffts. In mny rop plnts, s grmintion n rly sling growth r th most snsitiv stgs to nvironmntl strsss suh s slinity (Yilirim t l. 26; Khn n Pn 28). Slinity hs n shown to inrs th uptk of N or rs th uptk of C n K (Yilirim t l. 26). Spifi ion ffts my us irt toxiity or, ltrntivly, th insoluility or omptitiv sorption of ions my fft plnt nutritionl lns (Grnwy n Munns 198). Plnts xpos to slt strss pt thir mtolism in orr to op with th hnging nvironmnt. Survivl unr ths strssful onitions pns on th plnt s ility to priv th stimulus, gnrt n trnsmit signls n instigt iohmil hngs tht just mtolism oringly (Hsgw t l. 2). Slinity strss is known to triggr oxitiv strss in plnt tissus through th inrs in rtiv oxygn spis (ROS). Chloroplsts r th mjor orgnlls prouing ROS suh s suproxi rils, hyrogn proxi (H 2 O 2 ) n singlt oxygn uring photosynthsis (Apl n Hirt 24). Slt strss inus signifint rution in photosynthsis, whih pns on photosynthsizing tissu (lf r) n photosynthti pigmnts (Duy 2). ROS us hlorophyll (Chl) grtion n mmrn lipi proxition. So, mlonilhy (MDA) umultion s prout of lipi proxition n Chl rtntion r two oxitiv strss initors tht r tools for trmining slt tolrn in plnts (Yilirim t l. 28). To svng ROS, plnts possss spifi mhnisms, whih inlu tivtion of ntioxint nzyms n non nzymti ntioxints (Mittlr 22; Jll t l. 26). ROS triort mmrn funtion (Mishr n Chouhuri 1999); NO 3 - uptk n its rution wr limit to nitrogn ssimiltion n plnt growth unr slin onitions (Silvir t l. 21). To svng ROS, plnts synthsiz iffrnt typs of ntioxint ompouns or tivt ky ntioxint nzyms (Mittlr 22). Th llvition of oxitiv mg n inrs rsistn to slinity n othr nvironmnt strsss r oftn orrlt with n ffiint ntioxitiv systm (Jll t l. 27). Bing th worl s most svr prsiti inftion, mlri uss mor thn million ths n million ss nnully. Artmisinin, ssquitrpn lton ontining n noproxi rig n its rivtivs r fftiv ginst multi-rug rsistnt, Plsmoium fliprum strins minly in Southst Asi n mor rntly in Afri, without ny rput ss of rsistn (Krmsnr n Krishn 24). It is promising rug, s it hs lk of ross rsistn with othr nti-mlril rugs, no known vrs ffts to humns, n th ility to lr th loo of prsits mor rpily thn othr vill rugs (Mshnik t l. 1996). Sin 21 rtmisinin-s omintion thrpis (ACTs) r rommn y th Worl Hlth Orgniztion (WHO 26). This rommntion inrs th mn for rtmisinin, ling to supply shortgs, lthough th sitution hs n rntly rport s ing unr ontrol (Roll Bk Mlri 24). It is importnt to xplor nw mthos to nhn proutivity of this immnsly importnt rug plnt n lso to ut short its ost. Its hmil synthsis is possil ut is omplit n unonomilly vil u to poor yils (Ain t l. 23), thrfor th intt plnt rmins th only vil sour of th rtmisinin proution, Riv: 27 April, 21. Apt: Otor, 21. Originl Rsrh Ppr

2 Plnt Strss 4 (1), Glol Sin Books n th nhn proution of th rtmisinin ontnt in th whol plnt is highly sirl (Ain t l. 23; Aft t l. 21, 21, 211). Although thr r som rports rgring th rsponss of this immnsly importnt ntimlril plnt to slinity, stuis on th mging ffts of slt strss on growth n proutivity in trms of oxitiv strss n ntioxint nzyms r, howvr limit. Prs t l. (1998) foun tht th yil inrs signifintly with inrsing slinity strss, ut furthr inrss in slinity rs th yil. Th rtmisinin ontnt in th vgttiv tissu ws not influn with slinity strss. Qurshi t l. (2) n Qin t l. (27) rport tht th yil ws ngtivly influn y slt strss, howvr, rtmisinin ontnt inrs. Th urrnt stuy ws thrfor unrtkn to trmin th growth, photosynthti msurmnts, lipi proxition, ntioxint nzyms n hngs in rtmisinin ontnt ssoit with slinity strss. MATERIALS AND METHODS A grnhous pot xprimnt ws onut to nlyz th ffts of iffrnt onntrtions of soil slinity viz.,, 1, n 2 mm t th flowring stg ( ys ftr sowing) of A. nnu plnts. Slinity ws ppli whn th plnts wr t th stg of 3-4 tru lvs. To voi osmoti shok, NCl onntrtion ws inrs grully y 2 mm vry y, from zro, until th finl onntrtion of 2 mm ws rh. All th hmils us wr of nlytil gr n purhs from Sigm-Alrih, USA. Anlyss of growth n yil prmtrs Th plnts from h trtmnt wr uproot rfully n shoot hight ws ror. Plnts wr wsh with tp wtr to rmov hring forign prtils. Roots of th plnt wr rmov n frsh mss of th shoots ws ror iniviully. Th shoots wr ri t 8 C for 48 h, n ry mss ws thn ror. Totl lvs of th plnts wr wigh to trmin lf yil. Dtrmintion of photosynthti prmtrs n pigmnts Nt photosynthti rt (P N ) ws msur on sunny ys t 11: m using fully xpn lvs of A. nnu with th hlp of n IRGA (Infr R Gs Anlyzr, LI-COR 64 Portl Photosynthsis Systm, Linoln, Nrsk, USA). Bfor roring th msurmnt, th IRGA ws lirt n zro ws just pproximtly vry 3 min uring th msurmnt prio. Eh lf ws nlos in gs xhng hmr for 6 s. All th ttriuts msur y IRGA wr ror thr tims for h trtmnt. Totl Chl ontnt in frsh lvs ws stimt y th mtho of Lihtnthlr n Bushmnn (21). Th frsh tissu from young lvs ws groun using mortr n pstl ontining 8% ton. Th sorn of th solution ws ror t 662 n 64 nm for Chl stimtion using sptrophotomtr (Shimzu UV-17, Tokyo, Jpn). Assy of roni nhyrs tivity Croni nhyrs (CA; E.C ) tivity ws msur in frsh lvs using th mtho s sri y Dwivi n Rnhw (1974). 2 mg of frsh lf pis wr wigh n trnsfrr to Ptri ishs. Th lf pis wr ipp in 1 ml of.2 M ystin hyrohlori solution for 2 min t 4 C. To h tst tu, 4 ml of.2 M soium iront solution n.2 ml of.22% romothymol lu wr. Th rtion mixtur ws titrt ginst. N HCl using mthyl r s initor. Th nzym ws xprss s M CO 2 kg -1 lf FW s -1. Dtrmintion of ltrolyt lkg n prolin (Pro) ontnt with oul istill wtr (DDW) to rmov surf ontmintion. Young lf iss from h smpl wr tkn. Lf iss wr pl in los vil ontining 1 ml of DDW n inut on rottory shkr for 24 h. Susquntly th ltril onutivity of solution (EC 1 ) ws trmin. Smpls wr thn utolv t 12 C for 2 min n lst ltril onutivity (EC 2 ) ws not ftr ooling th solution t room tmprtur. Eltrolyt lkg ws lult s: Eltrolyt lkg (%) = (EC 1 /EC 2 ) 1 Th Pro ontnt ws stimt y th mtho of Bts t l. (1973). Th lvs wr homogniz in 3% quous sulfosliyli i n th homognt ws ntrifug t 1, rpm. Th suprntnt ws us for th stimtion of Pro ontnt. Th rtion mixtur onsist of 2 ml i ninhyrin n 2 ml of glil ti i, ws oil t 1 C for 1 h. Aftr trmintion of th rtion in n i th, th rtion mixtur ws xtrt with 4 ml of tolun n sorn ws r t 2 nm. Lipi proxition rt (TBARS ontnt) Oxitiv mg to lf lipis ws stimt y th ontnt of totl 2-thiorituri i rtiv sustns (TBARS) xprss s quivlnts of mlonilhy (MDA). TBARS ontnt ws stimt y th mtho of Ckmk n Horst (1991). TBARS wr xtrt from. g hopp frsh lvs, groun in ml of.1% (w/v) trihloroti i (TCA). Following th ntrifugtion t 12 g for min, 1-mL liquot from th suprntnt ws to 4 ml of.% (w/v) TBA in 2% (w/v) TCA. Smpls wr inut t 9 C for 3 min. Thrftr, th rtion ws stopp in n i th. Cntrifugtion ws prform t 1 g for min, n sorn of th suprntnt ws r t 32 nm on sptrophotomtr n orrt for non-spifi turiity y sutrting th sorn t 6 nm. TBARS ontnt ws xprss s nmol g 1 FW. Antioxint nzyms ssy Ctls (CAT; E.C ) tivity ws msur oring th mtho givn y Chnl n Snlios (1984) with smll moifition. Th ssy mixtur ontin 2.6 ml of mm potssium phospht uffr (ph 7.),.4 ml of mm H 2 O 2 n.4 ml of nzym xtrt. Th omposition of H 2 O 2 ws follow y lin in sorn t 24 nm. Enzym tivity ws xprss in U mg -1 protin (U = 1 mm of H 2 O 2 rution min -1 mg -1 protin). Proxis (POX; E.C ) tivity ws ssy y th mtho of Kumr n Khn (1982). Th ssy mixtur of POX ontin 2 ml of.1 M phospht uffr (ph 6.8), 1 ml of.1 M pyrogllol, 1 ml of. M H 2 O 2 n. ml of nzym xtrt. Th solution ws inut for min t 2 C ftr whih th rtion ws trmint y ing 1 ml of 2. N H 2 SO 4. Th mount of purpurogllin form ws trmin y msuring th sorn t 42 nm ginst rgnt lnk prpr y ing th xtrt ftr th ition of 2. N H 2 SO 4 t zro tim. Th tivity ws xprss in U mg 1 protin. 1 U of th nzym tivity orrspon to n mount of nzym tht us n inrs in th sorn of.1 min 1 mg 1 protin. Suproxi ismuts (SOD; E.C ) tivity ws ssy s sri y Buhmp n Friovih (1971). Th rtion mixtur ontin M rioflvin,.1 M mthionin, 2 1 M KCN n.6 1 M nitrolu ttrzolium slt (NBT) issolv in 3 ml of. M soium phospht uffr (ph 7.8) n 3 ml of th rtion mium ws to 1 ml of nzym xtrt. Th mixturs wr illumint in glss tst tus y two sts of Philips 4W fluorsnt tus in singl row. Illumintion ws strt to initit th rtion t 3 C for 1 h. Intil solutions tht wr kpt unr rk srv s lnks. Th sorn ws r t 6 nm in th sptrophotomtr ginst th lnk. SOD tivity ws xprss s U mg 1 protin. 1 U ws fin s th mount of hng in th sorn y.1 h 1 mg 1 protin. Eltrolyt lkg ws us to ssss mmrn prmility s sri y Lutts t l. (199). Smpls wr wsh thr tims 37

3 Slt strss-inu hngs in Artmisi nnu. Aft t l. Dtrmintion of nognous H 2 O 2 proution Th ontnt of H 2 O 2 in th lvs ws trmin oring to th mtho of Mukhrj n Chouhuri (1983). Frsh lvs of A. nnu (. g) wr homogniz using ol mortr n pstl in pr-ool ton ( ml) n th homognt ws ntrifug t 12 g for min. On ml of th suprntnt ws mix with.1 ml of % Ti(SO 4 ) 2 n.2 ml 19% mmoni. Aftr pripitt ws form, th rtion mixtur ws ntrifug t 12 g for min. Th rsulting pllt ws issolv in 3 ml of 2 M H 2 SO 4 n th sorn ws r t 4 nm using sptrophotomtr. Th H 2 O 2 onntrtion ws lult oring to stnr urv of H 2 O 2 rnging from to 1 M. Artmisinin xtrtion n stimtion Dry lf mtril (1 g) ws us to stimt rtmisinin moifi to ompoun Q 26 (rtmisinin ws onvrt to ompoun Q 26 s this is UV soring ompoun to tt on UV-ttor) n quntifi using HPLC mtho (Zho n Zng 1986). A stnr urv ws prpr using 1 mg of stnr rtmisinin issolv in 1 ml of HPLC-gr mthnol to mk th stok solution. It ws xtrt with 2 ml ptrolum thr in shkr t 7 rpm for 24 h. Aftr 24 h, th solvnt ws nt n pool n 2 ml of ptrolum thr gin n this stp ws rpt thr tims. Ptrolum thr frtions wr pool n onntrt unr ru prssur n rsius -ftt with CH 3 CN (1 ml 3). Pripitt ft ws filtr out n filtrt onntrt unr ru prssur. Rsius wr issolv in 1 ml of mthnol. 1 μl liquot of h smpl of h trtmnt ws tkn n to this 4 ml of.3% NOH ws. Th smpls wr inut in shking wtr th t C for 3 min, thrftr ool n nutrliz with glil ti i (.1 M in 2% MOH). Th ph of th solution ws mintin t 6.8. Drivtiz rtmisinin ws nlyz n quntifi through rvrs phs olumn (C18; μm; 4.6 mm; 2 mm) using prmix mthnol: 1 mm K-Phospht uffr (ph, 6.) in th rtio of 6:4 s moil phs t onstnt flow rt of 1 ml/min, with th ttor st t 26 nm. Artmisinin ws quntifi ginst th stnr urv of rtmisinin, otin from Sigm-Alrih, USA. Sttistil nlysis Eh pot ws trt s on rplit n ll th trtmnts wr rplit fiv tims. Th t wr nlyz sttistilly using SPSS-17 sttistil softwr (SPSS In., Chigo, IL, USA). Mn vlus wr sttistilly ompr y Dunn s multipl rng tst (DMRT) t P <.%. RESULTS Th prsn of slinity in th soil mium signifintly lowr th vlus for growth ttriuts (Shoot n root lngths, shoot n root ry wights). Shoot lngth in 2 mm NCl-trt plnts ws ru y 6.% ompr to ontrol plnts (Tl 1). Root lngth ws mor svrly fft y NCl toxiity ompr to shoots. In 2 mm NCl-trt plnts, root lngth ws ru 7.4% om- Tl 1 Efft of iffrnt slinity trtmnts on growth ttriuts of Artmisi nnu L. Mns within olumn follow y th sm lttr r not signifintly iffrnt ompr y Dunn s multipl rng tst (P.). Th t shown r mns of fiv rplits ± SE. Trtmnts Shoot lngth (m) Root lngth (m) Shoot ry mss (g) Root ry mss (g) Control 89.4 ± ± ± ±.83 mm NCl 84.1 ± ± ± ±.81 1 mm NCl 77.9 ± ± ± ±.72 mm NCl 67.3 ± ± ± ±.67 2 mm NCl.7 ± ± ± ±.4 Nt photosynthti rt (μ mol CO 2 m 2 s -1 ) 2 1 A B Stomtl onutn (mol m 2 s -1 ) Intrnl CO 2 (μ mol m -2 s -1 ) C D Totl hlorophyll ontnt ( mg g -1 FW). Control mm 1 mm mm 2 mm Control mm 1 mm mm 2 mm Slinity Trtmnts Slinity Trtmnts Fig 1 Efft of iffrnt slinity trtmnts on nt photosynthti rt (A), stomtl onutn (B), intrnl CO 2 (C) n totl hlorophyll ontnt (D) of Artmisi nnu L. Brs showing th sm lttr r not signifintly iffrnt t P. s trmin y Dunn s multipl rng tst. Th t shown r mns of fiv rplits n rror rs show SE. 38

4 Plnt Strss 4 (1), Glol Sin Books Croni nhyrs tivity [mol(co 2 ) kg -1 (FW) s -1 ] Eltrolyt lkg (%) A B oth y 143 n 1%, rsptivly ompr to th ontrol (Fig. 2B, 2C). Th vlus of TBARS ontnt n ntioxint nzyms signifintly nhn in th plnts sujt to slt strss. Th highst TBARS ontnt ws not whn th plnts wr suppli with 2 mm NCl through th soil (Fig. 3A). CAT tivity ws high in th plnts riving iffrnt NCl trtmnts, th most toxi fft ing t 2 mm NCl (Fig. 3B). Compr to th ontrol, th tivity of POX n SOD lso signifintly inrs in slt-trt plnts n th pplition of 2 mm NCl to A. nnu plnts show highst tivity of POX whil SOD tivity ws mximum t mm of NCl (Fig. 3B, 3C). Th ontnt of nognous H 2 O 2 ws msur in orr to trmin th intrnl ROS sttus y slt strss in A. nnu plnts. H 2 O 2 ontnt ws highr in plnts trt with n 1 mm NCl, ompr to th ontrol, suggsting n inrmnt in th lvl of ROS in lls. At 1 mm of slt, th inrs in intrnl H 2 O 2 ws 44.9% ompr to th ontrol (Fig. 4A). Th rtmisinin ontnt ws highst whn 1 mm NCl ws ppli through soil (47.8% mor) ompr to untrt plnts, lthough rs in rtmisinin ontnt ws not whn highr oss of slt wr ppli (Fig. 4B). DISCUSSION Prolin ontnt (mg g -1 FW) 1 pr to untrt plnts (Tl 1). Shoot n root ry wights wr lso signifintly ru y iffrnt slinity trtmnts. At 2 mm NCl (th highst ppli onntrtion), th shoot n root ry wights of th plnt wr ru y 82.1 n 77.8%, rsptivly, ompr to th ontrol (Tl 1). A ru rt of photosynthti tivity ws osrv in th slinity trt plnts. In omprison to ontrol, t 2 mm NCl onntrtion, th nt photosynthti rt ws 98.4% lowr showing th gr of mg to th lf tissus (Fig. 1A). Both, stomtl onutn n intrnl CO 2 ws osrv th lowst (111.8 n 7.7% lss thn th ontrol, rsptivly) t 2 mm NCl (Fig. 1B, 1C). Chl ontnt ws lso ru in slt-strss plnts n th most toxi fft ws not t 2 mm NCl t whih th ontnt ws 87.9% lss thn tht of untrt plnts (Fig. 1D). Slinity strss rs th tivity of CA msur in th lvs of trt plnts; t 2 mm NCl, th tivity of CA ws ru y 116% ompr to th ontrol (Fig. 2A). Th toxi fft of slinity on ltrolyt lkg n Pro ws proportiont to th onntrtion ppli n th highst ppli onntrtion (2 mm NCl) rs Control mm 1 mm mm 2 mm Slinity Trtmnts Fig. 2 Efft of iffrnt slinity trtmnts on CA tivity (A), ltrolyt lkg (B) n prolin ontnt (C) of Artmisi nnu L. Brs showing th sm lttr r not signifintly iffrnt t P. s trmin y Dunn s multipl rng tst. Th t shown r mns of fiv rplits n rror rs show SE. C Growth prmtrs From th rsults of th prsnt stuy, it is lr tht slinity us n ovrll rution in growth of A. nnu plnts whn ompr with th ontrol (Tl 1), whih is in grmnt with wht hs n rlir rport in iffrnt rops (Athr t l. 28; Siiqui t l. 28; Khn t l. 21). Prs t l. (1998) foun tht th vgttiv yil inrs signifintly with inrsing slinity strss to 6. S/m, ut furthr inrss in slinity rs th yil. Qurshi t l. (2) n Qin t l. (27) rport tht th yil ws ngtivly influn y slt strss. Slinity rus th imiition of wtr us of lowr osmoti potntils of th mium n uss hngs in mtoli tivitis, thry ling to rution in growth (Mnguzzo t l. 1999; Jll t l. 27). Also, slt strss limits plnt growth y vrsly ffting vrious physiologil n iohmil prosss, inluing ntioxint pity n homostsis (Ashrf 24). Rution in growth of A. nnu in rspons to inrsing slt strss might hv n u to vrition in numr of iohmil or physiologil trits tht r ssoit with mhnisms of slt tolrn suh s photosynthsis, nutrint homoostsis, umultion of omptil soluts n tivitis of ntioxint nzyms, t. Photosynthti prmtrs Extnsiv litrtur is vill whih shows tht plnt growth is pnnt on plnt photosynthti pity. Thrfor, lin in proutivity us of slinity strss is oftn ssoit with rution in photosynthsis pity (Ashrf 24; Duy 2). In th prsnt stuy, ru nt photosynthti rt, stomtl onutn n intrnl CO 2 onntrtion ws noti whn plnts wr xpos to slinity. Slt strss mgs th photosynthti mhinry t multipl lvls, suh s pigmnts, stomtl funtioning n gsous xhng, strutur n funtion of thylkoi mmrn, ltron trnsport n nzyms (Suhir n Murthy 24). Exss slt uss stomt to los, thry rsing th prtil CO 2 prssur (Bthky n Drw 1992) s wll s intrnl CO 2 onntrtion n onsquntly rs in th nt photosynthti rt. In numr of stuis with iffrnt rop spis, slt strss ru photosynthti rt (Ashrf 24; Khn t l. 21). As osrv in th prsnt stuy, oxitiv strss might inu u to rs stomtl onutn in rspons to ioti strss. Consquntly, u to limittion of CO 2, 39

5 Slt strss-inu hngs in Artmisi nnu. Aft t l. 2 A B 2 TBRAS ontnt (nmol g -1 FW) 1 1 CAT tivity (Unit mg -1 protin) 1 C D 2. POX tivity (Unit mg -1 protin) SOD tivity (Unit mg -1 FW) 2. Control mm 1 mm mm 2 mm Slinity Trtmnts Control mm 1 mm mm 2 mm Slinity Trtmnts Fig. 3 Efft of iffrnt slinity trtmnts on TBRAS ontnt (A), CAT (B), POX (C) n SOD (D) tivitis in lvs of Artmisi nnu L. Brs showing th sm lttr r not signifintly iffrnt t P. s trmin y Dunn s multipl rng tst. Th t shown r mns of fiv rplits n rror rs show SE.. A B 8 H 2 O 2 ontnt (nmol g -1 FW ) Artmisinin ontnt (μg/g DW) Control mm 1 mm mm 2 mm Slinity Trtmnts Control mm 1 mm mm 2 mm Slinity Trtmnts Fig. 4 Efft of iffrnt slinity trtmnts on H 2O 2 (A) n rtmisinin (B) ontnt of Artmisi nnu L. Brs showing th sm lttr r not signifintly iffrnt t P. s trmin y Dunn s multipl rng tst. Th t shown r mns of fiv rplits n rror rs show SE. ltrons from PSII r vill to form ftty is of thylkois n strom (Qurshi t l. 2). Th possil isruption of thylkoi n stomtl mmrns rsults in hlorosis n nrosis ling ultimtly to rs in photosynthsis n photosynthti pigmnts, hn ru vilility of photosynthts for iomss umultion. Th rs in Chl ontnt n ttriut to oxition of Chl n hloroplsti mmrns; instility of th pigmnt protin omplx, whih might xrt y xss slt, s rport y Stpin n Klous (26) in Cuumis stivus on pplying 1 mm of slt. Ths osrvtions of lowr Chl ontnt r onsistnt with thos of Shim t l. (23) n Stpin n Klous (26) who init tht Chl ontnt onsirly rs in th lvs of spinh n uumr plnts, rsptivly with inrsing NCl onntrtions from to 1 mm. Pri n Ds (2) rport tht slt strss inhiit th Chl ontnt in th lvs of mny rops. 4

6 Plnt Strss 4 (1), Glol Sin Books Physiologil n iohmil prmtrs CA is th nzym tht plys mny ivrs rols in physiologil prosss suh s ion xhng, i s ln, roxyltion/roxyltion rtions n inorgni ron iffusion twn th ll n its nvironmnt s wll s within th ll (Gorgios t l. 24). Also, th tivity of CA, whih tlyzs th intronvrsion of CO 2 n HCO 3, is rgult y photon flux nsity, CO 2 onntrtion, vilility of Zn (Tiwri t l. 2) n xprssion of gns noing CA protin (Kim t l. 1994). NCl-f plnts show th lowst CA tivity, whih my u to intivtion of Ruiso, whih squntilly rus th nt photosynthti rt, ron mtolism, lf Chl ontnt n photosynthti ffiiny (Smn n Crithly 198). Eltrolyt lkg nls ll mmrn injury to ssss whn plnts r sujt to slinity strss. Mintining intgrity of llulr mmrns unr slt strss is onsir n intgrl prt of slinity tolrn mhnism (Stvns t l. 26). Th plnts trt with NCl xhiit signifint inrs in ltrolyt lkg ompr to th ontrol plnts. It hs n rport tht slt strss l to signifint inrs in th lvl of ltrolyt lkg in mny rop (Pri n Ds 2; Khn t l. 27; Siiqui t l. 28). A omptil solut whih umults unr slt strss in plnts is Pro. In th prsnt stuy, n inrs in Pro ontnt in th NCl-trt plnts ws not, ing highst t 2 mm (Fig. 2C). Although th pris rol of Pro umultion is still t, it is oftn onsir s omptil solut involv in osmoti justmnt (Azooz t l. 24). Th umultion of Pro my through n inrs in its synthsis onstntly with inhiition of its tolism (Yoshi t l. 1997) n my mhnism for strss tolrn. Howvr, its rol in imprting strss rsistn unr slin onitions is ontrovrsil. Anywy, unrstning th iosynthsis, grtion, trnsport n rol of Pro uring strss n th signlling vnts tht rgult strss-inu umultion is vitl in vloping plnts for strss tolrn (Jll t l. 27). Th lipi proxition rt (TBARS ontnt) ws msur in lvs s n initor of oxitiv strss. Inrmnt in oxitiv strss ws not whn th plnts wr xpos to iffrnt onntrtions of slinity (Fig. 3A). Mittlr (22) propos tht mmrn mg might us y high H 2 O 2 lvls, whih oul lrt th formtion of hyroxyl ril n thus lipi proxition. In NCltrt plnts, oxitiv strss might inu u to th rs stomtl onutn in rspons to th osmoti imln n ru lf wtr potntil. Lipi proxition hs n ssoit with mgs provok y vrity of nvironmntl strsss (Hrnànz t l. 23). Th inrs in lipi proxition n orrlt with th umultion of ions n ROS proution unr slt strss (Hrnànz t l. 23; Misr n Gupt 26). In orn with th prsnt rsults, othrs hv foun tht xss slt inrs TBARS ontnt in A. nnu n othr plnts (Qurshi t l. 2; Pn t l. 26; Jll t l. 27, 28). Plnts with high lvls of ntioxints, ithr onstitutiv or inu, hv n rport to hv grtr rsistn to oxitiv mg (Suhkr t l. 21). Environmntl strsss inrs th formtion of ROS tht oxiiz mmrn lipis, protin n nuli is (Gong t l. 2). Unr slt strss, plnts r ovrlo with ROS, whih inhiit svrl plnt prosss n us mg to th plnts in iffrnt wys. ROS gnrt hyroxyl rils n othr strutiv spis suh s lipi proxis (Viynthn t l. 23), whih uss strution of ll mmrn s rflt y inrs vlus for TBARS unr slt strss. Thus, to mintin mtoli funtions unr strss, th svnging of ROS is rquir. ROS svnging pns on toxifition mhnism provi y ntioxint nzyms. CAT n POX tivitis inrs signifintly in NCl-trt plnts with rspt to th ontrol, whil SOD tivity inrs up to mm NCl n thrftr rs in its tivity ws not (Fig. 3B-D). An inrs in CAT, POX n SOD, ws oumnt y mny othrs invstigting th slinity strss in svrl plnt spis (Ko t l. 27; Pn t l. 26; Jll t l. 27, 28). CAT n POX ppr to ply n ssntil prottiv rol in svnging pross of ROS whn oorint with SOD (Jll t l. 29). SOD initits toxifition of singlt oxygn y forming H 2 O 2, whih is lso toxi n must limint y onvrsion to H 2 O in susqunt rtions. In plnts, numr of nzyms rgult intrllulr H 2 O 2 lvls, ut CAT n POX r onsir th most importnt (Notor n Foyr 1988). Suproxi rils r toxi y-prouts of oxitiv mtolism n n intrt with H 2 O 2 to form highly rtiv hyroxyl rils, whih r thought to primrily rsponsil for oxygn toxiity in th ll (Azvo t l. 2). Th ismuttion of suproxi rils into H 2 O 2 n oxygn is n importnt stp in protting th ll n is tlyz y SOD. Th H 2 O 2 onntrtion ws inrs signifintly y pplying n 1 mm NCl n thrftr rs in th onntrtion of H 2 O 2 ws osrv (Fig. 4A). A ommon nomintor in ll vrs onitions (ioti strsss) is th ovr proution of ROS within iffrnt llulr omprtmnt of th plnt ll (Pinhiro t l. 24). Enhn ROS proution in A. nnu ws tt y othrs u to iffrnt nvironmntl strsss (Qurshi t l. 2; Frrir 27; Guo t l. 21). In our prvious stuy, w rport tht mil oron strss (. n 1. mm) inrs th H 2 O 2 proution in A. nnu plnts (Aft t l. 21). As CAT, POX n SOD r known to svngrs of ROS, whn th ontnt of ROS rhs ov th thrshol, thy svng thm n th osrv rution in th ontnt of H 2 O 2 ftr rtin NCl onntrtion is ovious. Artmisinin ontnt n yil Prs t l. (1998) foun tht th rtmisinin ontnt in th vgttiv tissu ws not influn with slinity strss whil Qurshi t l. (2) n Qin t l. (27) rport tht th rtmisinin ontnt inrs on slinity strss. An ssoition twn H 2 O 2 n rtmisinin proution ws not in th prsnt stuy. Ovrproution of rtmisinin ontnt ws osrv whn n 1 mm NCl ws ppli n t furthr high onntrtions it ws rs (Fig. 4B). Wllrt t l. (2), Frrir (27), Pu t l. (29) n Guo t l. (21) show irt rltionship twn ROS n rtmisinin ontnt. In our l stuy, w osrv tht rtmisinin ontnt nhn signifintly upon xposur to oron strss (. n 1. mm) n rs y pplying highr onntrtions (Aft t l. 21). From th urrnt rsrh it is lr tht low lvls of slinity ( n 1 mm) promot th iosynthsis of rtmisinin n lso w hv stlish th possil onntion twn rtmisinin n H 2 O 2, whih is onsir to ply n importnt rol of onvrting ihyrortmisini i to rtmisinin uring rtmisinin synthsis. Wllrt t l. (1999) suggst tht ihyrortmisini i might t s svngr of ROS tht r rls in plnt lls whn thy r xpos to oxitiv strss. Our rsults r in grmnt with th osrvtions ror y Wllrt t l. (2), Frrir (27), Pu t l. (29) n Guo t l. (21) who vot tht rltivly high lvls of ROS wr prou on ourrn of vrious strsss tht in turn, rsult in nhn onvrsion of ihyrortmisini i to rtmisinin. REFERENCES Ain MZ, Isrr M, Rhmn RU, Jin SK (23) Artmisinin, novl ntimlril rug: Biohmil n molulr pprohs for nhn proution. Plnt Mi 69, 1-11 Aft T, Khn MMA, Irs M, Nm M, Singh M, Rm M (21) Stimultion of rop proutivity, photosynthsis n rtmisinin proution in Artmisi nnu L. y triontnol n girlli i pplition. Journl of Plnt Intrtions, Aft T, Khn MMA, Irs M, Nm M, Rm M (21) Boron inu 41

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8 Plnt Strss 4 (1), Glol Sin Books Wllrt TE, Prs N, Bkmn AC, Qux WJ (2) Ssonl vrition of rtmisinin n its iosynthti prursors in plnts of Artmisi nnu of iffrnt gogrphil origin: proof for th xistn of hmotyps. Plnt Mi 66, 7-62 Wllrt TE, vn Un W, Lurink HG, Worng HJ, Prs N, Qux WJ (1999) Isoltion n intifition of ihyrortmisini i from Artmisi nnu n its possil rol in th iosynthsis of rtmisinin. Journl of Nturl Prouts 62, WHO (26) WHO Guilins for th Trtmnt of Mlri, Worl Hlth Orgniztion Yilirim E, Tylor AG, Spittlr TD (26) Amliortiv ffts of iologil trtmnts on growth of sqush plnts unr slt strss. Sinti Hortiultur 111, 1-6 Yilirim E, Turn M, Guvn I (28) Efft of folir sliyli i pplitions on growth, hlorophyll n minrl ontnt of uumr (Cuumis stivus L.) grown unr slt strss. Journl of Plnt Nutrition 31, Yoshi Y, Kiyous T, Nkshim K, Ymguhi-Shinozki K, Shinozki K (1997) Rgultion of lvls of prolin s n osmolyt in plnts unr wtr strss. Plnt n Cll Physiology 38, Zho SS, Zng MY (1986) Dtrmintion of qinghosu in Artmisi nnu L. y high prformn liqui hromtogrphy. Chins Journl of Phrmutil Anlysis 6, 3-43