Differential Responses of the Activities of Antioxidant Enzymes to Thermal Stresses between Two Invasive Eupatorium Species in China

Transcription

1 Journl o Intgrtiv Plnt Biology 28 Dirntil Rsponss o th Ativitis o Antioxint Enzyms to Thrml Strsss twn Two Invsiv Euptorium Spis in Chin Ping Lu 1,2, Wi-Guo Sng 1 n K-Ping M 1 ( 1 Ky Lortory o Vgttion n Environmnt Chng, Institut o Botny, th Chins Amy o Sins, Bijing 193, Chin; 2 Rsours n Environmntl Sins Collg, Northst Agriulturl Univrsity, Hrin 153, Chin) Astrt Th t o thrml strss on th ntioxint systm ws invstigt in two invsiv plnts, Euptorium nophorum Sprng. n E. oortum L. Th ormr is snsitiv to high tmprtur, whrs th lttr is snsitiv to low tmprtur. Our im ws to xplor th rltionship twn th rspons o ntioxint nzyms n tmprtur in th two invsiv ws with irnt istriution pttrns in Chin. Plnts wr trnsrr rom glsshous to growth hmrs t onstnt 25 C or 1 wk to limtiz to th nvironmnt. For th ht trtmnts, tmprtur ws inrs stpwis to 3, 35, 38 n inlly to 42 C. For th ol trtmnts, tmprtur ws rs stpwis to 2, 15, 1 n inlly to 5 C. Plnts wr kpt in th growth hmrs or 24 h t h tmprtur stp. In E. nophorum, th oorint inrs o th tivitis o ntioxint nzyms ws tiv in protting th plnt rom th umultion o tiv oxygn spis (AOS) t low tmprtur, ut th tivitis o tls (CAT), guiol proxis (POD), sort proxis (APX), glutthion ruts (GR), n monohyrosort ruts (MDAR) wr not ompni y th inrs o suproxi ismuts (SOD) uring th ht trtmnts. As rsult, th lvl o lipi proxition in E. nophorum ws highr unr ht strss thn unr ol strss. In E. oortum, howvr, th lssr gr o mmrn mg, s init y low monohyrosort ontnt, n th oorint inrs o th oxygn. Dtoxiying nzyms wr osrv in ht-trt plnts, ut th ntioxint nzyms wr unl to oprt in ol strss. This inits tht th plnts hv highr pity or svnging oxygn rils in ht strss thn in ol strss. Th irnt rsponss o ntioxint nzyms my on o th possil mhnisms o th irns in tmprtur snsitivitis o th two plnt spis. Ky wors: ntioxint nzym systm; ol strss; Euptorium nophorum; Euptorium oortum; ht strss; invsiv plnts. Lu P, Sng WG, M KP (28). Dirntil rsponss o th tivitis o ntioxint nzyms to thrml strsss twn two invsiv Euptorium spis in Chin. J. Intgr. Plnt Biol. oi: /j x. Avill onlin t Euptorium nophorum Sprng. n Euptorium oortum L. r prnnil mmrs o th Composit mily. Sin thir introution to Chin in th rly 2th ntury, oth spis hv om invsiv psts (Ding 22; Li 22), ut thir gogrphil istriutions in Chin r irnt (Figur 1). Riv 15 Jn. 27 Apt 2 Mr. 27 Support y Ntionl Nturl Sin Fountion o Chin (347337), Knowlg Innovtion Projt o Chins Amy o Sins (KSCX1- SW-13-3) n K C Wong Fllowship, Royl Soity o UK. Author or orrsponn. Tl: +86 () ; Fx: +86 () ; E-mil: <swg@is..n>. C 28 Institut o Botny, th Chins Amy o Sins oi: /j x E. oortum, ntiv to Cntrl n South Amri, is olsnsitiv n is t tmprturs low C (Wu n Xu 1992). Hn, its istriution hs n rstrit to southrn Chin, n its northwr spr is slowr thn E. nophorum. In ontrst, E. nophorum, ntiv to Cntrl Amri, oul surviv in th tmprtur rng ( C) (Zho n M 1989), n xpn to northrn Chin muh str thn E. oortum. A s stuy init tht th rt o spr ws out 6 km pr yr rom south to north in Chin (Xing 1991). Howvr, E. oortum is ttr l thn E. nophorum to tolrt high tmprturs. E. oortum inv th hot, ry vllys o Yunnn n Sihun provins whr tmprturs r high (mximum tmprtur 4.4 C, Co 1993) n whih r rltivly ri. E. nophorum is rrly oun in th hot, ry vllys. Th irns o th two lin spis in istriution n invsiv potntil pos intrsting qustions onrning th mhnisms tht trmin thir vntul istriution.

2 2 Journl o Intgrtiv Plnt Biology 28 Figur 1. Distriutions o Euptorium nophorum n E. oortum in Chin. E. nophorum ( ); E. oortum ( ). Howvr, thr hs n littl rsrh on thir physiologil n iohmil mhnisms tht onr irnt ht n/or ol rsistn. A ttr unrstning o ths mhnisms is vry importnt or ontrolling n priting th potntil istriutions o th two spis. It hs n provn tht th rspons o ntioxint nzym systms is on o th most importnt mhnisms o nvironmntl limtiztion or plnts. Th rol o ths nzym systms or mking plnts tolrnt to xtrm nvironmnts hs lry n monstrt (Dhins n Mtow 1981; Shnr n Krus 199; Hrnánz t l. 2). It is urrntly ssum tht th ngtiv t o vrious nvironmntl strsss is t lst prtilly u to th gnrtion o tiv oxygn spis (AOS) n/or th inhiition o th systm tht ns ginst thm (As 1997; Alshr t l. 1997). AOS r highly rtiv n, in th sn o ny prottiv mhnism, n sriously isrupt norml mtolism through oxiting mmrn lipis, protin n nuli is (Smirno 1993; Foyr t l. 1994, 1994). Howvr, to mitigt n rpir mg initit y AOS, plnts volv llulr ptiv rsponss suh s uprgultion o oxitiv strss prottors n umultion o prottiv soluts (Horling t l. 23). Antioxint ns nzyms suh s suproxi ismuts (SOD), tls (CAT), sort proxis (APX), guiol proxis (POD), glutthion ruts (GR), monohyrosort ruts (MDAR) n hyrosort ruts (DHAR) r systms tht minimiz th onntrtions o suproxi n hyrogn proxi. Thror, in this ppr, w ompr th rspons o th ntioxint nzym systm to ht n ol strsss in E. nophorum n E. oortum. Spiilly, w stui hngs in lipi proxition n th ntioxint nzyms SOD, CAT, POD, APX, GR, MDAR n DHAR unr ht n ol strss. Rsults n Disussion Suproxi ismuts tlyss th ismuttion o O 2 to H 2 O 2 n O 2 (Bowlr t l. 1992). Both spis xhiit n inrs in SOD tivity in oth ht n ol strsss (Figur 2A, D). In lvs o E. nophorum, th 38 C n 42 C trtmnts us 61% n 138% inrs in totl SOD tivity in omprison to th ontrol plnts, rsptivly, n th highst totl SOD tivity ws osrv t 42 C. In lvs o E. oortum, howvr, th highst totl SOD tivity ws osrv t 5 C. SOD hs lso n rport to inrs on xposur to ht strss in mulrry (Chitny t l. 22), n to ol strss in ly spurg (Dvis n Swnson 21). Ctls limints H 2 O 2 y rking it own irtly to orm wtr n oxygn. In lvs o E. nophorum, CAT tivity ws prss y high tmprtur, n inrs to high lvl in th low tmprtur trtmnt (Figur 2B). In lvs o E. oortum, howvr, th rspons pttrn o CAT tivity ws rvrs, CAT tivity ws nhn y high tmprtur n th plnts wr unl to mintin norml tivity whn sujt to low tmprtur, suh s 1 C n 5 C (Figur 2E). An inrs in SOD tivity with rs in CAT tivity hs n rport whn th plnts wr sujt to ioti strss (Sruym n Tni 1995; Fu n Hung 21; Jung 23). Othrs hv oun tht CAT tivity

3 Antioxint Enzyms Rspon to Ht in Euptorium Spis 3 E. nophorum E. oortum Suproxi ismuts (Units/mg protin) A Suproxi ismuts (Units/mg protin) D Ctls tivity (µmol H 2 O 2 /min pr mg protin) B Ctls tivity (µmol H 2 O 2 /min pr mg protin) E Proxis tivity (µmol H 2 O 2 /min pr mg protin) C Proxis tivity (µmol H 2 O 2 /min pr mg protin) F Tmprtur ( C ) Tmprtur ( C ) Figur 2. Et o tmprtur on suproxi ismuts (SOD), tls (CAT) n guiol proxis (POD) tivitis o Euptorium nophorum (A C) n E. oortum (D F). Vlus r th mns o thr rplits with stnr rrors. Mns with th sm lttrs r signiintly irnt t P.5 using Dunn s multipl rng tst. is photointivt unr low n high tmprtur strsss n CAT tivity vris with plnt spis (Firn t l. 1992). Guiol proxis tlyss H 2 O 2 -pnnt oxition o sustrt. In lvs o E. nophorum, POD tivity inrs rmrkly with rsing tmprturs rom 25 Cto5 C, nling th plnts to prott thmslvs ginst th oxitiv strss, ut in ht strss, POD tivity ws lmost th sm until th tmprtur ws t 38 C, n thn lin (Figur 2C). In ontrst, in lvs o E. oortum, POD tivity ws signiintly nhn y th ht strss, ut in ol strss, POD tivity rmin unhng until th tmprtur ws t 5 C, n thn rs (Figur 2F). Ths rsults wr onsistnt with thos o Rivro t l. (21), who rport irntil rsponss o POD tivity to thrml strss twn tomto n wtrmlon plnts. In wtrmlon plnts, POD tivity inrs unr high tmprtur, ut rs unr low tmprtur. In tomto plnts, POD tivity rs unr high n low tmprtur, n th lowst POD tivity ws oun unr high tmprtur strss. Asort proxis uss sort s th ltron onor or th rution o H 2 O 2 n is wll known to importnt in th toxiition o H 2 O 2. In lvs o E. nophorum, th tivity o APX inrs with rsing tmprtur rom

4 4 Journl o Intgrtiv Plnt Biology 28 E. nophorum E. oortum Asort proxis tivity (µmol sort/ min pr mg protin) A Asort proxis tivity (µmol sort/ min pr mg protin) D Glutthion ruts tivity (µmol NADPH/min pr mg protin) Monohyrosort ruts (µmol NADH/min pr mg protin) B C Glutthion ruts tivity (µmol NADPH/ min pr mg protin) Monohyrosort ruts (µmol NADH/min pr mg protin) E F g Tmprtur ( C ) Tmprtur ( C ) Figur 3. Et o tmprtur on sort proxis (APX), glutthion ruts (GR) n monohyrosort ruts (MDAR) tivitis o Euptorium nophorum (A C) n E. oortum (D F). Vlus r th mns o thr rplits with stnr rrors. Mns with th sm lttrs r signiintly irnt t P.5 using Dunn s multipl rng tst. 25 Cto5 C, ut in ht strss, th tivity o APX rh pk vlu t 3 C n 35 C n thn lin (Figur 3A). In lvs o E. oortum, howvr, th tivity o APX ws inrs y inrsing th tmprtur rom 25 Cto42 C, ut in ol strss, th tivity o APX rh pk vlu t 15 C n thn lin (Figur 3D). Th rsti rss o APX tivitis t 38 Cor42 C in lvs o E. nophorum, suggst tht APX n not ompnst or th loss o CAT n POD tivitis t high tmprturs, whih sms to svrly ru th H 2 O 2 -svnging pity. Howvr, uring ol trtmnt, roton w xhiit n inrs tivity o H 2 O 2 toxiying nzym APX, CAT n POD. Thus, th lvls o mmrn lipi proxition wr rltivly lowr unr ol strss, whih sms to rlt to its poor ht tolrn. Th rstrition o roton w to tmprt limts (minly on th Yunnn-Guizhou Pltu) (Lu n M 24) suggsts tht poor ht-strss rsistn my tor limiting th istriution o th plnts. Whil in E. oortum, th rsti rss o APX

5 Antioxint Enzyms Rspon to Ht in Euptorium Spis 5 tivitis wr oun t 1 Cor5 C, whih sms to rlt to its poor ol tolrn. Glutthion ruts, nothr nzym in th As-Hlliwll pthwy, n lso rmov ytosoli hyroproxis suh s lipi hyroproxi, using ru glutthion s o-sustrt (Y n Grssl 2). In lvs o E. nophorum, th tivity o GR ws inrs signiintly y rsing th tmprtur rom 25 Cto5 C, ut in ht strss, th GR tivity ws prss (Figur 3B). In lvs o E. oortum, howvr, th tivity o GR ws inrs y inrsing th tmprtur rom 25 Cto42 C, ut in ol strss, th tivity o GR ws unhng (Figur 3E). Aoring to Foyr n Hlliwll (1976), H 2 O 2 is minly limint y th sort-glutthion yl. Bsis th svnging t o CAT n POD, ytotoxiity o H 2 O 2, whih might prou u to onstitutiv n inu lvls o SOD in oth spis, might ispos o with th i o oth APX n GR. Th sort-glutthion yl ns MDAR n DHAR in ition to APX n GR. MDAR n DHAR r nzyms rsponsil or sort rgnrtion in plnt tissus. Th tivity o MDAR rs signiintly in ht strss, whrs MDAR tivity in lvs o th ol-trt E. nophorum ws not signiintly irnt rom th ontrols (Figur 3C). In lvs o E. oortum, howvr, th tivity o MDAR ws inrs y inrsing th tmprtur rom 25 Cto42 C, ut in ol strss, th tivity o MDAR ws rs (Figur 3F). Dhyrosort ruts tivity in lvs o th two spis inrs with oth ht n ol strsss (Figur 4A, C). In oth spis, grtr inrs in DHAR tivity ws oun with ht trtmnt thn in ol trtmnt, n th highst DHAR tivity ws ror t 42 C. DHAR is thought to ply n importnt rol in th oxitiv strss tolrn o plnts y rgnrting sort rom hyrosort (Foyr n Mullinux 1998). Th uprgultion o DHAR tivity in oth ol-snsitiv n ht-snsitiv, n ol-tolrnt n httolrnt invsiv plnts rthr thn MDHAR, suggsts tht in E. nophorum n/or E. oortum sujt to thrml strss onitions, sort is rgnrt vi glutthion. It my tht DHAR tivity oul prtiipt in sort rgnrtion unr onitions o svr strss whn MDHAR tivity is E. nophorum E. oortum Dhyrosort ruts MDA ontnt (nmol/g FW) (µmol sort/min pr mg protin) A B Dhyrosort ruts (µmol sort/min pr mg protin) MDA ontnt (nmol/g FW) C D i h g g Tmprtur ( C ) Tmprtur ( C ) Figur 4. Et o tmprtur on hyrosort ruts (DHAR) tivity n Lipi proxition o Euptorium nophorum (A, B) n E. oortum (C, D). Vlus r th mns o thr rplits with stnr rrors. Mns with th sm lttrs r signiintly irnt t P.5 using Dunn s multipl rng tst.

6 6 Journl o Intgrtiv Plnt Biology 28 limit y th vilility o NADH (As n Tkhshi 1987; Hrnnz t l. 2). This is in ontrst to th sitution sri y Jhnk t l. (1991) who rport th DHAR losing lmost ll o its tivity in ol strss o two Z gnotyps iring in ol tolrn, Jin t l. (23) who rport th tivitis o DHAR wr not snsitiv to hngs in tmprtur o iv onirs with irnt ol tolrn. Ht n ol strsss l to signiint inrs in MDA ontnt in oth spis (Figur 4B, D). MDA umultion in E. nophorum lvs ws grtr with ht trtmnt thn with ol trtmnt, with grtr inrs t 42 C thn t 5 C, initing tht highr gr o lipi proxition ourr t highr tmprturs. In lvs o E. oortum, howvr, th MDA umultion ws grtr with ol trtmnt thn with ht trtmnt, with grtr inrs t 5 C thn t 42 C, initing highr gr o lipi proxition inu y ol strss. Exposur to low tmprtur my inrs th mount o tiv oxygn spis not only in ol-snsitiv E. oortum, ut lso in ol-tolrnt plnts lik E. nophorum, lthough E. nophorum show lvt tivitis o SOD, CAT, POD, APX, GR, MDAR n DHAR on xposur to ol trtmnt, th pility o toxiition o AOS is limit. This is similr to som rports tht hv monstrt th lvt tivitis o ntioxint nzyms in slt-tolrnt ri vritis whil th lvl o lipi proxition ws inrs unr onitions o slinity strss (Viynthn t l. 23). Th intrtions o APX, CAT, SOD n thir involvmnt in svnging AOS r vry omplx, n lso involv othr proxiss (Snlios 1997, 1997; Notor n Foyr 1998). Th xtnt o oxitiv strss using mmrn n llulr mg might possily ir pning on th strss impos. Plnts my rspon to irnt nvironmntl strsss in irnt wys, s shown in our xprimnt (Figurs 2 4). SOD tlyzs th ismuttion o suproxi into oxygn n hyrogn proxi, so th inrs o SOD tivity suggst n inrs proution o H 2 O 2.H 2 O 2 is limint y tls n proxiss, whih inlu oth nzymti n non-nzymti H 2 O 2 grtion (Pltzr t l. 22). Ctls omposs H 2 O 2 into wtr, whrs POD omposs H 2 O 2 y oxition o o-sustrts suh s phnoli ompouns n/or ntioxints (Blokhin t l. 23). Th ntioxints suh s sort n glutthion r involv in svnging H 2 O 2 primrily vi th Hlliwll-As pthwy (Hormns t l. 2). It hs n suggst tht hyrogn proxi must tivly svng in orr to minimiz ytotoxiity y tivt oxygn in th prsn o lvt SOD tivity (Finzzi-Agro t l. 1986; Sot t l. 1987). In E. nophorum, th oorint inrs o oxygntoxiying nzyms ws tiv in protting th plnt rom th umultion o AOS t low tmprturs, thus vrting llulr mg unr unvorl onitions. Howvr, th tivitis o CAT, POD, APX, GR n MDAR wr not ompni with inrs SOD uring ht strss; thus in ht-trt E. nophorum, ytotoxiity ws likly u to th umultion o H 2 O 2, whih n rt with O 2 to prou hyroxyl r rils vi th Hrrt-Wiss rtion (Bowlr t l. 1992). As rsult, th lvl o lipi proxition in E. nophorum ws highr unr ht strss thn unr ol strss. In E. oortum, howvr, th lssr gr o mmrn mg (s init y low MDA ontnt) n th oorint inrs o th oxygn-toxiying nzyms ws osrv in ht-trt plnts, ut th ntioxint nzyms wr unl to o this with ol strss. This inits tht th plnt hs highr pity or svnging oxygn rils in ht strss thn in ol strss. Th ivrs rspons o ntioxint nzyms suh s SOD n othr nzyms (CAT, POD, APX, MDAR, GR) u to thrml strss suggsts th rol o oxitiv strss s omponnt o nvironmntl strss on th two invsiv spis. It pprs tht th irns in SOD n othr nzyms (CAT, POD, APX, MDAR, GR) hs irt rltion to th snsitivity o th two plnts to tmprtur. In ri, inrs tivity o SOD n rs tivitis o CAT n POD wr tt in ol-snsitiv ultivrs, whrs inrs tivitis o ths nzyms hv n oun in rsistnt ultivrs xpos to hilling strss, whil GR in oth ultivrs ws stl to low tmprtur strss (Sruym n Tni 1995). Clr irns wr oun in th ol snsitivity o CAT n APX twn th ol-snsitiv n ol-tolrnt ri ultivrs. In miz, th tivity o SOD n APX o th rsistnt vrity o plnts grown t low tmprturs wr highr thn in ontrols. In ontrst, in th snsitiv vrity o plnts grown t low tmprtur, th tivity o SOD ws highr thn in th ontrols n th tivity o APX ws lowr thn in th ontrols (Mssi t l. 1995). Our rsults suggst tht oorint inrs o th tivity o oxygn-toxiying nzyms oul nssry to prott E. nophorum lvs rom th umultion o oxygn rils t low tmprturs, or prott E. oortum lvs rom th umultion o oxygn rils t high tmprturs. Although svrl nvironmntl strsss in plnts r known to inu th xprssion n/or inrsing lvls o ntioxitiv nzyms n thir mrnas (Pn n Yu 1992; Klinstin t l. 1998; Morit t l. 1999), th mhnism y whih ths tivt oxygn-svnging nzyms n work o-oprtivly, in rspons to tmprtur strss is not yt known. Furthr nlysis o th rgultion o gn xprssion in ths nzyms shoul luit th mhnism o irnt tmprtur tolrns. Intiying th gns tht rgult som o th nzyms involv in strss n ining wys to intrr with gn rgultion my prov usul to iing n onomilly vil ontrol o prsistnt prnnil ws in iiult-to-rh lotions or long wtrwys whr th us o hriis is unsirl or illgl (Dvis n Swnson 21). Our utur gol lso involvs xmining th long-trm rsponss o th ntioxitiv systm, to urthr luit thir untion in thrml tolrn mhnisms o th two invsiv spis.

7 Antioxint Enzyms Rspon to Ht in Euptorium Spis 7 Consiring th irnt istriution pttrns o E. nophorum n E. oortum in Chin, our t suggst tht th irns o ntioxint nzym tivitis n MDA ontnt in th two spis my sri to irns in mhnisms unrlying oxitiv strss injury n susqunt tolrn to tmprtur strss. Mtrils n Mthos Plnt mtril n ultur onitions Euptorium nophorum Sprng. slings wr ollt rom th Kunming Institut o Botny, th Chins Amy o Sins n E. oortum L. slings wr ollt rom th Xishungnn Tropil Botnil Grn, th Chins Amy o Sins. Slings wr plnt in plsti pots (19 m imtr n 16 m high) ill with 1:1:1 mixtur o soil:sn:pt. Thy wr grown in glsshous with y/night tmprturs o 3 C/2 C. Mximl photosynthti photo lux nsity ws pproximtly 8 µmol/m 2 pr s. Plnts wr wtr ily n rtiliz on rgulr sis. Atr 4wks, whn th plnts o oth spis h hiv sty stt in thir growth n rh hights o out 3 4 m, thy wr sujt to th tmprtur trtmnts. Ht n ol strss trtmnts Plnts o o h invsiv spis wr ivi into two groups. For th ht strss trtmnt, on hl o th plnts wr mov rom th glsshous n pl in growth hmrs t onstnt tmprtur o 25 C n rltiv humiity o 7%, 12/12 h light/rk photoprio rgim, n photon lux nsity o 25 µmol/m 2 pr s or 7 ys to limtiz th plnts to th growth hmrs. Atr limtiztion, th tmprtur ws inrs stpwis to 3 C, 35 C, 38 C n inlly to 42 C n xpos to h tmprtur t 24 h intrvls. Simultnously, or th ol strss trtmnt, th sm limtiztion prio o 7 ys ws us. Anothr group o plnts wr thn xpos to progrssivly rsing tmprturs o 2 C, 15 C, 1 C n 5 C t 24 h intrvls. Eh trtmnt ws rpt in thr sprt growth hmrs. Th soil ws kpt sturt t il pity uring oth trtmnts to voi possil rought. Th plnts tht wr kpt t 25 C or 24 h wr us s ontrols. Atr h 24 h tmprtur trtmnt, ully xpn l mtril ws ollt rom thr plnts pr trtmnt, rozn, stor in liqui nitrogn n mintin t 7 C. Th tissu ws groun n th nzyms xtrt or ssy. Lipi proxition Lipi proxition in E. nophorum n E. oortum ws trmin y msuring th mlonilhy (MDA) ontnt, prout o lipi proxition, in 1 g l rsh wight ollowing Mhv Ro n Srsty (2). Enzym xtrtion On grm o rozn l mtril ws homogniz in 8 ml o 5 mm phospht ur (ph 7.) tht inlu 1 mm thylniminttrti i (EDTA) n 2% (w/v) polyvinylpolypyrrolion (PVP). Th homognt ws ntriug t 13 g or 4 min t 4 C. In th ss o APX n DHAR tivitis trmintion, 1 mm sort n 2 mm 2-mrptothnol, rsptivly, wr to th homognizing ur to prvnt nzym intivtion. Th suprntnt ws us or ssys o nzym tivity n protin ontnt. Totl solul protin ontnts o th nzym xtrts wr trmin ollowing Bror (1976), using ovin srum lumin (BSA) s stnr. Enzym ssys Suproxi ismuts tivity ws trmin using th mtho o Buhmp n Friovih (1971) tht msurs th inhiition in th photohmil rution o nitrolu ttrzolium (NBT) sptrophotomtrilly t 56 nm. Msuring th tivity o CAT ollow Knörr t l. (1996) y monitoring th rt o omposition o H 2 O 2 t 24 nm or 2 min. Msuring th tivity o POD ollow Chn n Mhly (1955) y monitoring th rt o th inrs in th sorn t 47 nm or 2 min u to guiol oxition. Th tivity o APX ws msur y ollowing Nkno n As (1981), n monitoring th rt o sort oxition t 29 nm or 3 min. Th tivity o GR ws ssy oring to th mtho o Hlliwll n Foyr (1978) y ollowing th rs in sorn t 34 nm or 3 min u to NADPH oxition. Th nzym tivity o MDAR ws msur in th suprntnt t 25 C s sri y Hossin t l. (1984). MDAR ws ssy y ollowing th rs in sorn t 34 nm or 5 min u to NADH oxition. Th tivity o DHAR ws msur in th suprntnt t 25 C s sri y Hossin n As (1984) y monitoring th inrs in sorn t 265 nm or 3 min u to sort ormtion. Sttistil nlysis Th xprimnt ws rrng in ompltly rnomiz lok sign with thr rplits. Dunn s multipl rng tst or multipl omprisons ws us or th nlysis o sttistil signiin. Comprisons with P vlus.5 wr onsir signiintly irnt. All vlus rport r mns ± SE o thr rplits. Aknowlgmnts Mny thnks to Mrk Willimson, Yulong Fng n F. Anrw Smith or thir suggstions out this ppr n or giving muh

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