Ox i da tive stress in her bi cide-treated broad bean and maize plants

Nemat M. Hassan and Mamdouh M. Nemat Alla

Bot any De part ment, Fac ulty of Sci ence at Damietta, Mansoura Uni ver sity, Damietta, Egypt

Fax: +20.57.403868 E-mail: mamnematalla@mum.mans.edu.eg.

Key words: broad bean, her bi cide, maize, ox i da tive stress

Ab stract

Treat ments of broad bean and maize seed lings with fluo -meturon, atrazine or rimsulfuron af fected some pa ram e ters ofox i da tive stress. Fluometuron sig nif i cantly re duced ac tiv ity ofHill re ac tion (PSII), chlo ro phyll a+b con tents and ac tiv ity ofribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco,EC 4.1.1.39) in leaves of both spe cies and sig nif i cantly in -creased con tents of H2O2, lipid per ox ides and car bonyl groupsdur ing the whole ex per i ment. There were, more over, sig nif i -cant in hi bi tions in ac tiv i ties of superoxide dismutase (SOD;E.C. 1.15.1.1), catalase (CAT; E.C. 1.11.1.6), ascorbate per -oxidase (APX; E.C. 1.11.1.11) and guaiacol peroxidase (GPX; E.C. 1.11.1.7). Re sponse to atrazine was, to some ex tent, sim i -lar to fluometuron through out the en tire ex per i ment in broadbean and up mostly to the 12th day of the ex per i ment in maize.The her bi cide ef fect was more pro nounced in broad bean thanmaize. These re sults point to in di cate an oc cur rence of ox i da -tive stress in both spe cies by fluometuron and only in broadbean by atrazine. The in crease in H2O2 con tent ac com pa niedwith drop in ac tiv i ties of SOD, CAT and peroxidases in di catesa de cline in its de tox i fi ca tion rather than in crease in its syn the -sis. On the con trary, rimsulfuron seemed to have no ef fect onmost of the tested pa ram e ters al though there were tran sient sig -nif i cant in creases in H2O2, lipid per ox ides and car bonylgroups as well as ac tiv i ties of SOD, CAT, APX and GPX.These find ings, based on the re cov ery in ox i da tive stress, in di -cate that fluometuron is in volved in ox i da tive stress gen er a tion in both spe cies but atrazine only in broad bean whilerimsulfuron is not in both spe cies.

In tro duc tion

Plants, as other aer o bic or gan isms, re quire ox y genfor the ef fi cient pro duc tion of en ergy. Dur ing there duc tion of ox y gen to wa ter, ac tive ox y gen spe -cies (AOSs) such as superoxide rad i cals (O.-

2), hy -dro gen per ox ide (H2O2) and hydroxyl rad i cals(OH.-) can be formed. Among other AOSs, sin gletox y gen (1O2) is also ca pa ble of dam ag ing im por -tant plant cell con stit u ents (Foyer et al. 1994). Sin -glet ox y gen sources in bi o log i cal sys tems in cludelight-de pend ent re ac tions and chem i cal gen er a -tions in dark re ac tions (Briviba et al. 1997, Gulnerand Dodge 2000, Wakabayashi and Boger 2004).The neg a tive ef fect of stress is of ten me di ated byAOSs-ini ti ated ox i da tive dam age (Kuzniak 2002).Un der op ti mal en vi ron men tal con di tions, the an ti -ox i dant sys tem in plant cells ef fec tively pro tectsthem from po ten tially del e te ri ous ef fects of AOSs.How ever, un der en vi ron men tal stress con di tions,AOSs gen er a tion is en hanced, thus the cel lu lar an -ti ox i dant ca pac ity can be over-whelmed and ox i da -tive stress oc curs (Hippeli and Elstner 1996,Pasqualini et al. 2001, Noctor and Foyer 1998,Hernández et al. 2000). AOSs re act with lipids, pro -teins, pig ments and nu cleic ac ids and cause lipidperoxidation, mem brane dam age, in ac ti va tion ofen zymes, thus af fect ing cell vi a bil ity. Some her bi -cides pro duce ox i da tive stress (Van Camp et al.1994). The phenylurea her bi cide fluometuron

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[l,l-dimethy1-3 -(α,α,α-tri fluoro-m-tolyl)urea] and the S- tr iazine her bi c ide atrazine[ 2 - c h l o r o - 4 - e t h y l a m i n o - 6 - i s o p r o p y l -amino-1,3,5-triazine] con trols weeds and grassesby block ing the elec tron flow be tween the photo -systems (Kirkwood 1987, Dodge 1994). On theother hand, the sulfonylurea her bi cide rimsul furon[1-(4,6-dimethoxypyrimidin -2 -y1) -3-(py ri dylsul -fonyl)urea] acts by in hib it ing aceto hydroxy acidsynthase [AHAS, EC 4.1.3.18], the first en zyme invaline, leucine, and isoleucine biosynthesis (Mar -tinetti et al. 1995, Jiménez et al. 1997). Ex cess elec -trons are do nated to mo lec u lar ox y gen to gen er atesuperoxide rad i cals. Super oxide dismutase (SOD;E.C. 1.15.1.1) cat a lyzes the con ver sion of super -oxide rad i cals to H2O2 and O2. Hy dro gen per ox ideis re duced to wa ter by the ac tion of catalase (CAT;E.C. 1.11.1.6), or by ascor bate peroxidase (APX;E.C. 1.11.1.11) and guaiacol peroxidase (GPX;E.C. 1.11.1.7). There are ba sic dif fer ences in darkphase of pho to syn the sis be tween broad bean (C3)and maize (C4) plants. How ever, some other com -mon pa ram e ters in both spe cies were checked to in -ves ti gate their in ter fer ence by a given her bi cide.There fore, this work was aimed to study the dif fer -en tial ef fects of her bi cides be long ing to dif fer entgroups, on some phys i o log i cal parameters ofoxidative stress in broad bean and maize toelucidate which of these herbicides is possiblyinvolved in the generation of oxidative stress in C3and/or C4 plants.

Ma te ri als and Meth ods

Plant ma te ri als and growth con di tions

Broad bean (Vicia faba L., Giza 2) and maize (Zeamays L., hy brid 351) seeds were sur face ster il izedand ger mi nated in sand/clay soil (1/1, v/v) in pots(25 cm di am e ter x 20 cm height). Pots, af ter be ingleft in dark for 3 days, were trans ferred to a 14-hphotoperiod with 450-500 µmol·m-2·s-1 photo syn -thetic pho ton flux den sity, 75-80 % rel a tive hu mid -ity, and 22/10 °C or 28/14 °C day/night re gime forbroad bean or maize, re spec tively. When the seed -lings were ten days old, the pots were di vided intofour groups; one to serve as a con trol and one foreach her bi cide treat ment at a dos age equiv a lent tothe field rates (2.98, 1.79 and 0.015 kg ai·ha-1 for

fluometuron, atrazine and rimsulfuron, re spec -tively). The her bi cide treat ments were ap plied onlyonce. The rate per hect are was cal cu lated ac cord ing to the sur face area per pot and then the her bi cidequan tity was solubilized in a suit able amount ofwa ter. Seed lings in each pot were sprayed twice, inone di rec tion and cross wise with out avoid ing thatthe sprays reach the soil. Shoots were col lected justbe fore her bi cide ap pli ca tion (zero time) and alsoaf ter 2, 4, 8, 12, 16 and 20 days from the treat ment.Sam ples were washed with co pi ous amounts of wa -ter and blot ted dry with pa per tow els be fore thesub se quent anal y ses.

De ter mi na tion of Hill re ac tion ac tiv ity

and chloro phylls con tent

Leaf tis sues were ground in 50 mM Na-tricine(N-tris hydroxymethyl methyl glycine, pH 7.8), 0.3 M su crose and 3 mM MgCl2. The re sult ing ho mog -e nate was cen tri fuged at 2,000 x g for 10 min. Thepel lets were re sus pend ed in 0.1 M NaCl and thencen tri fuged again at 5,000 x g for 5 min utes. The re -sult ing pel lets were re sus pend ed in 1 mM Natricine (pH 7.8), 10 mM NaCI and 10 mM MgCI2.Photo system II ac tiv ity was mon i tored at 600 nm in 200 mM Na-phos phate (pH 6.7), 2 mM MgCl2 and0.5 mM 2,6-dichlorophenolindophenol (DCPIP),as in di cated by the rate of 2 ,6-DCPIPphotoreduction (Trebst 1972).

Con tents of chloro phylls were de ter mined in thefresh tis sues af ter ex trac tion with 85 % ac e tone ac -cord ing to the spec tro pho to met ric method de -scribed by Metzner et al. (Metzner et al. 1965).

As say of (Rubisco, EC 4.1.1.39)

Ac cord ing to Keys and Parry (1990), leaf tis sueswere ho mog e nized in 20 mM Tris-HCl (pH 8.0), 10 mM NaHCO3, 10 mM MgCl2, 1 mM EDTA, 5 mMdithiothreitol (DTT), 0.08 mM chlorohexidinediacetate, and 1 % (w/v) acid-washed in sol u blepolyvinylpyrrolidone. The ho mog e nate was cen tri -fuged at 5,000 x g for 15 min and the supernatantwas treated with solid (NH4)2SO4 to give 35 % sat -u ra tion. Af ter 20 min, the sus pen sion was cen tri -fuged at 20,000 x g for 15 min and the supernatantwas raised to 55 % sat u ra tion. Af ter centrifugationat 20,000 x g for 15 min, the pel let was dis solved in

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a so lu tion con tains 20 mM Tris, 1 mM DTT, 1 mMMgCl2 and 0.08 mM chlorohexidine diacetate andad justed at pH 8. Rubisco ac tiv ity was as sayed in50 mM 4-(2-hydroxymethyl)1-piperazine eth anesul fo nic acid (HEPES, pH 7.8), 10 mM NaHCO320 mM MgCl2 , 0 .66 mMribulose-1,5-bisphosphate (RuBP), 0.2 mMNADPH, 5 mM ATP, 5 mM creatine phos phate, 2.0U creatine phosphokinase , 2 .8 Uglyceraldehyde-3-phos phate dehydrogenase, and2.0 U phosphoglycerate kinase. The re ac tion wasstarted by ad di tion of the en zyme, and the con tentswere al lowed to re act at 30 °C for 30 min, dur ingwhich the de crease in absorbance at 340 nm wasmea sured. One unit was de fined as the ox i da tion ofµmol NADPH per min ute

De ter mi na tion of lipid peroxidation and car bonyl

groups in pro teins

Lipid peroxidation was de ter mined by mea sur ingthe con cen tra tion of thiobarbituric acid- (TBA-) re -act ing sub stances as de scribed by Buege and Aust(1972). Plant tis sues were ho mog e nized in 150 mM KCl, cen tri fuged at 7,000 x g for 15 min and 1 ml ofthe supernatant was in cu bated at 37 °C for 2 h af terwhich 1 ml of 0.6 M trichloroacetic acid was added. Af ter thor ough mix ing, the re ac tion mix ture wascen tri fuged at 5,000 x g for 10 min. One ml ofsupernatant was taken with an equal vol ume of2-thiobarbituric acid and placed in a boil ing wa terbath for 10 min, cooled, and di luted with 1 ml dis -tilled wa ter. Spec tro pho to met ric read ings at 535nm were cal cu lated as nmoles malonaldehyde(MDA)·g-1 plant tis sue and then con verted to nmolMDA·mg-1 pro tein.

Car bonyl groups were as sayed us ing the dinitro -phenyl hydrazine method (Le vine et al. 1991). Pro -teins were ex tracted from 0.5 g of plant tis sue with100 mM po tas sium phos phate (pH 7.0), 0.1 % Tri -ton X-100, 1 mM Na2EDTA. Af ter pre cip i ta tion ofthe pos si ble con tam i nat ing nu cleic ac ids in thesam ple with 1 % (w/v) strep to my cin sul fate, analiquot of 0.8 ml of the ex tract was re acted with 0.2ml of 20 mM dinitrophenyl hydrazine (in 2 M HCl)for 1 h, with vig or ous shak ing. Pro teins were thenpre cip i tated with 0.6 M TCA, washed with 1:1(v/v) eth a nol:ethyl ac e tate, solubilized in 6 Mguanidi ne-HCl (pH 4.5) and the absorbance of the

hy dra zones (derivatized carbonyls) was measuredat 370 nm.

De ter mi na tion of H2O2

Plant tis sues were ho mog e nized in 200 mM per -chloric acid, cen tri fuged at 5,000 x g for 10 min andthe supernatant was neu tral ized with 4 M KOH. Af -ter centrifugation at 3,000 x g for 5 min, 0.2 ml ofthe supernatant was loaded on 1 ml col umn ofDowex 1X8-100 an ion ex change resin and elutedwith 0.8 ml of dis tilled wa ter. Ac cord ing to Okudaet al. (1991), the as say mix ture con tained 1 ml ex -tract, 0.4 ml 12.5 mM 3-dimethylaminobenzoicacid in 375 mM phos phate buffer pH 6.5, 0.08 ml1.3 mM 3-methyl-2-benzothiazolinone hydrazone, and 0.02 ml (0.25 units) horse rad ish peroxidase.The re ac tion was ini ti ated by the ad di tion of peroxi -dase and the in crease in absorbance at 590 nm wasmonitored for 3 min.

As say of superoxide dismutase

(SOD; E.C. 1.15.1.1)

SOD ac tiv ity was as sayed by us ing the pho to chem -i cal nitroblue tetrazolium (NBT) method in termsof SOD’s abil ity to in hibit re duc tion of NBT toform formazan by superoxide (Beyer andFridovich 1987). Plant tis sues were ho mog e nizedin 50 mM phos phate, pH 7.8, 0.1 % (w/v) BSA, 5.5mM ascorbate, and 8 mM β-mercaptoethanol. SOD was as sayed in 50 mM phos phate, pH 7.8, 9.9 mML-methionine, 0.057 mM NBT, 0.025% (w/v) Tri -ton X-100, and 0.1 mM ri bo fla vin. The photore -duction of NBT (for ma tion of pur ple formazan)was mea sured at 560 nm and an in hi bi tion curvewas made against dif fer ent vol umes of ex tract. Oneunit of SOD was de fined as that be ing pres ent in thevol ume of ex tract that caused in hi bi tion of thephotoreduction of NBT by 50 % of con trol (0.025ab sorp tion units at 560 nm·min-1).

As say of catalase (CAT; E.C. 1.11.1.6)

Catalase ac tiv ity was de ter mined by mea sur ingH2O2-de pend ent ox y gen evo lu tion at room tem -per a ture with an ox y gen elec trode unit. Plant tis -sues were ho mog e nized in 50 mM po tas sium phos -phate buffer, pH 7, and 1 mM DTT. Catalase ac tiv -ity was as sayed in 50 mM phos phate buffer, pH 7

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OX I DA TIVE STRESS OF HER BI CIDE-TREATED PLANTS

and 33.5 mM H2O2 by mon i tor ing the pro duc tionof dioxy gen us ing an ox y gen elec trode (del Rio etal. 1977). One unit was de fined as the de com po si -tion of µmol H2O2 per min ute.

As say of ascorbate peroxidase

(APX; E.C. 1.11.1.11)

Plant tis sues were ho mog e nized in 0.1 M Tricine --KOH buffer, pH 8, 1 mM DTT, 10 mM MgCl2, 50mM KCl, 1 mM EDTA, 0.1 % (w/v) Tri ton X-100,and 0.28 mM phenylmethylsulfonyl flouride(PMSF) and cen tri fuged at 12,000 x g for 30 min at4 °C. The re ac tion me dium con tained 0.5 mMascor bic acid, 0.1 mM H2O2, 1 mM EDTA, and 0.1mM HEPES-KOH buffer, pH 7.8 (Ranieri et al.1996). The en zyme ac tiv ity was de ter mined fol -low ing the de crease in absorbance at 290 nm in thefirst 30 s from the start of the re ac tion us ing the ex -

tinc tion co ef fi cient of 2.8 mM-1·cm-1 for ascorbate.One en zy matic unit is equiv a lent to 1 µmol ofascor bic acid ox i dized·min-1.

As say of guaiacol peroxidase

(GPX; E.C. 1.11.1.7)

Plant tis sues were ho mog e nized in 220 mMTris-HCl, pH 7.4, 250 mM su crose, 50 mM KCl, 1mM MgCl2, 160 mM β-mercaptoethanol, and 0.57mM PMSF and cen tri fuged at 12,000 x g for 30 min at 4 °C. The re ac tion mix ture con tained 20 mM Naac e tate, pH 5, 30 mM H2O2, 2 mM guaiacol andplant ex tract. The rate of guaiacol ox i da tion was re -corded at 470 nm and the ac tiv ity was cal cu latedus ing the ex tinc tion co ef fi cient of 26.6 mM-1·cm-1

for tetraguaiacol (Ranieri et al. 1997). Theperoxidase ac tiv ity was con verted to peroxidaseunits us ing horse rad ish peroxidase stan dards andas sayed un der iden ti cal con di tions as sam ples.

Pro tein con tent was de ter mined spec tro pho to met -ri cally by re ac tion with Commassie Bril liant BlueG ac cord ing to Brad ford (1976). All val ues re -ported herein are means (±SD) of at least six bi o -log i cal rep li ca tions from two in de pend ent ex per i -ments. The full data were sta tis ti cally an a lyzed us -ing the least sig nif i cant dif fer ence (LSD) test at 5 % level (Snedecor and Cochran 1980).

Re sults and Dis cus sion

Fig ure 1 rep re sents the changes in the ac tiv ity ofHill re ac tion (Photosystem II) of broad bean andmaize leaves, as in di cated by the rate of photore -duction of 2,6-dichlorophenol indophenol(2,6-DCPIP). As com pared with con trols, fluome -turon in duced sig nif i cant re duc tion in the rates ofHill re ac tion ac tiv ity in both spe cies through out theex per i ment. Atrazine, on the other hand, sig nif i -cantly re duced Hill re ac tion ac tiv ity in broad beandur ing the whole ex per i ment and in maize up to the12th day of treat ment, there af ter the her bi cide be -came of no sig nif i cant ef fect. The greater re duc tion

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Fig. 1. Ef fect of her bi cides on 2,6-dichlorophenolindophenol(2,6-DCPIP) photoreduction, chlo ro phyll a+b con tents andRubisco ac tiv ity in leaves of broad bean and maize. The datapre sented are means (±SD) of at least six bi o log i cal rep li ca -tions from two in de pend ent ex per i ments. Ver ti cal bars rep re -sent LSD at 5 % level.

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in Hill re ac tion ac tiv ity due to fluometuron oratrazine was ob served in broad bean than in maize.On the con trary there were no changes at all in Hillre ac tion ac tiv ity in leaves of both spe cies as a re sultof treat ment with rimsulfuron dur ing the en tire ex -per i men tal pe riod.

In ad di tion, fluometuron sig nif i cantly and pro gres -sively de creased the con tents of chlo ro phyll a+b ofbroad bean and maize seed lings dur ing the en tireex per i ment as com pared with the re spec tive con -trols (Fig. 1). The mag ni tude of de crease aug men -ted with the elapse of time. Atrazine in hib ited chlo -ro phyll ac cu mu la tion in broad bean dur ing thewhole ex per i ment and in maize only dur ing the first 12 days of treat ment, there af ter no sig nif i cant dif -fer ences from con trol were found. The de crease inchlo ro phyll a+b by ei ther her bi cide with re spect tocon trol was greater in broad bean than in maize. Onthe other hand, rimsulfuron had no sig nif i cant ef -fects on chlo ro phyll a+b of both spe cies al though a

sig nif i cant de crease was observed in broad beanonly during the first 4 days of treatment.

The ob served de pres sion in chlo ro phyll con tentsand the ac tiv ity of Hill re ac tion in both spe cies inre sponse to treat ment with fluometuron, and tosome ex tent with atrazine, ap pears to co in cide witha de crease in ac tiv ity of Rubisco, the key en zyme in photosynthetic ef fi ciency (Fig. 1). The Fig ureshows that the ac tiv ity of Rubisco in shoots of bothspe cies was sig nif i cantly and con sis tently in hib ited by fluometuron treat ment dur ing the en tire ex per i -

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Fig. 4. Ef fect of her bi cides on superoxide dismutase ac tiv ity inleaves of broad bean and maize. The data pre sented are means(±SD) of at least six bi o log i cal rep li ca tions from two in de -pend ent ex per i ments. Ver ti cal bars rep re sent LSD at 5 % level.

Fig. 3. Ef fect of her bi cides on H2O2 con tents in leaves of broad bean and maize. The data pre sented are means (±SD) of at least six bi o log i cal rep li ca tions from two in de pend ent ex per i ments.Ver ti cal bars rep re sent LSD at 5 % level.

Fig. 2. Ef fect of her bi cides on lipid peroxidation prod ucts andcar bonyl groups con tents in leaves of broad bean and maize.The data pre sented are means (±SD) of at least six bi o log i calrep li ca tions from two in de pend ent ex per i ments. Ver ti cal barsrep re sent LSD at 5 % level.

men tal pe riod as com pared with con trol val ues ateach in ter val. The en zyme in hi bi tion in creasedwith time. Atrazine slowed down the ac tiv ity in -crease ob served in the con trols. In com par i son tocon trol lev els at each in ter val, atrazine re sulted in asig nif i cant in hi bi tion in the en zyme ac tiv ity ofbroad bean dur ing the whole ex per i ment and ofmaize up to the 12th day of treat ment. The mag ni -tude of in hi bi tion in ac tiv ity of the en zyme ac tiv itywas higher in broad bean than in maize. The ef fectof rimsulfuron on the en zyme ac tiv ity was in sig nif -i cant from the 2nd day of treatment onward either inbroad bean or in maize.

Dur ing pe ri ods of se vere stress, the in creased rateof AOSs pro duc tion re sults in dam age to the photo -synthetic ap pa ra tus (Van Breusegem et al. 1999). In ac cor dance, PSI and PSII were de creased in manyplant spe cies in con se quence to sev eral stress stim -uli (Allakhverdiev et al. 2000, El-Shahaby et al.2002). There fore, AOSs gen er a tion due to the dis -tur bances in the elec tron trans port rate of PSI andPSII could sub se quently cause deg ra da tion ofphoto synthetic pig ments and in hi bi tion of theirbiosynthesis with a con se quence de crease in pho to -syn the sis. In ad di tion, some en zymes of photosyn -thetic car bon re duc tion cy cle could also be re spon -si ble for pho to syn the sis de creases. Of the im por -tant en zymes in driv ing pho to syn the sis in both C3and C4 plants, Rubisco be ing the most es sen tialone; it is by far the most abun dant pro tein pres ent in

green leaves of higher plants (Spreitzer 1993).Vecchia et al (2001) re ported that bleach ing her bi -cides caused se vere photo-ox i da tive dam age tomaize chloroplasts. They, more over, stated that un -like C3 spe cies, C4 spe cies can ex press a strong tol -er ance to her bi cides. This sug ges tion could sup port the pres ent ob ser va tions that fluometuron andatrazine had more se vere ef fects on broad bean than in maize. On the other hand, Kingston-Smith andFoyer (2000) found that paraquat treat ment tomaize mod i fies PSII re ac tion cen ter polypeptide,D1, in creased the num ber of polypeptides aris ingfrom break down of Rubisco and in creased car -bonyl groups in bun dle sheath. These ef fects mightre sult from the gen er ated AOSs that would re actwith lipids, pro teins and pig ments caus ing lipidperoxidation and mem brane dam age. Con se -quently, the in creased ac cu mu la tions of lipidperoxides and carbonyl groups due to lipidperoxidation and protein oxidation respectivelywere checked.

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Fig. 5. Ef fect of her bi cides on catalase ac tiv ity in leaves ofbroad bean and maize. The data pre sented are means (±SD) ofat least six bi o log i cal rep li ca tions from two in de pend ent ex per -i ments. Ver ti cal bars rep re sent LSD at 5 % level.

Fig. 6. Ef fect of her bi cides on ascorbate peroxidase andguaiacol peroxidase ac tiv i ties in leaves of broad bean andmaize. The data pre sented are means (±SD) of at least six bi o -log i cal rep li ca tions from two in de pend ent ex per i ments. Ver ti -cal bars rep re sent LSD at 5 % level.

Fig ure 2 clearly in di cates that lev els of TBA- re act -ing sub stances and car bonyl groups pro duc tion in -creased in broad bean and maize fol low ing her bi -cide treat ments. The great est in crease in MDA andcar bonyl groups oc curred in fluometuron-treatedplants in both spe cies dur ing the en tire pe riod of the ex per i ment. Atrazine also re sulted in sig nif i cant in -creased lipid per ox ides and car bonyl groups pro -duc tion with re spect to con trol val ues ei ther inbroad bean through out the whole ex per i ment or inmaize only dur ing the first 12 days of treat ment.The ef fect of fluometuron and atrazine on the ac cu -mu la tion of MDA and car bonyl groups was greaterin broad bean than in maize. Rimsulfuron treat ment to both spe cies in duced slight in creases in both pa -ram e ters only at the start in com par i son to con trolval ues. In broad bean and maize, lipid per ox ides in -creased sig nif i cantly dur ing the first 8 and 4 days of treat ment, re spec tively and carbonyl groups duringthe first 4 and 2 days, respectively.

The in creased rate of MDA and car bonyl groupsgives an in dex of lipid peroxidation and pro tein ox -i da tion and there fore, of ox i da tive stress. In creas -ing in TBA-re act ing sub stances were ob served inmany plant spe cies due to sev eral fac tors (Dixit etal. 2001, Sandalio et al. 2001). More over,Halliwell and Gutteridge (1989) con firmed that theperoxi dation of cell mem branes se verely af fects itsfunc tion al ity and in teg rity and can pro duce ir re -vers ible dam age to the cell func tion. On the otherhand, Reinheckel et al. (1998) re ported that thefunc tion al ity of pro teins can be af fected by ROSsei ther by ox i da tion of amino acid side chains or bysec ond ary re ac tions with al de hy dic prod ucts oflipid peroxi dation. Both pri mary and sec ond ary re -ac tions can in tro duce car bonyl groups into pro -teins. Such con di tions of ox i da tive stress can alsobe en hanced by a de crease of en zy matic an ti ox i -dants in volved in the de tox i fi ca tion of AOSs.These en zymes cat a lyze the for ma tion and scav -eng ing of H2O2 which is con sid ered a po tent re ac -tive sub stance that can in ac ti vate en zymes by ox i -diz ing their thiol groups (Bowler et al. 1994).

In Fig ure 3, H2O2 con tent in broad bean showed asig nif i cant in crease fol low ing treat ment withfluometuron or atrazine dur ing the whole ex per i -men tal pe riod and with rimsulfuron dur ing the first8 days of treat ment. In maize, H2O2 con tent was

sig nif i cantly higher in fluometuron-treated sam -ples than in con trols dur ing the whole ex per i ment.Atrazine and rimsulfuron sig nif i cantly in creasedthese con tents only dur ing the first 8 and 4 days oftreat ment, re spec tively. The mag ni tude of in creasein H2O2 con tent in treated sam ples was more ap par -ent in broad bean than in maize. How ever, therewere no sig nif i cant changes rel a tive to con trols ei -ther in atrazine-treated broad bean or in both spe -cies treated with rimsulfuron. The for ma tion ofH2O2 or its scav eng ing might de pend on the en -zymes SOD or CAT, APX and GPX, re spec tively.Therefore, the activities of these enzymes werechecked.

SOD ac tiv ity was sig nif i cantly in hib ited in broadbean treated with ei ther fluometuron or atrazinedur ing the en tire ex per i ment as well as in maizetreated with fluometuron (Fig. 4). The in hi bi tion ofSOD by fluometuron at the end of the ex per i mentwas greater in broad bean than in maize. In con trast, in maize treated with atrazine the ac tiv ity of SODsig nif i cantly in creased up to the 12th day of treat -ment. A sim i lar re sponse was ob served by rimsul -furon up to the 8th and the 4th day of treat ment inbroad bean and maize, re spec tively and then itdecreased to control levels.

A re duc tion in SOD ac tiv ity due to fluometuronand atrazine might be at trib uted to in ac ti va tion ofthe en zyme by H2O2 that is pro duced in dif fer entcel lu lar com part ments where SOD ca tal y ses thedisproportionation of superoxide rad i cals (Bowleret al. 1994, Van Camp et al. 1994, 1996, Sandalio et al. 2001). More over, en hanced lev els of SOD inplants has been cor re lated with tol er ance to ox i da -tive stress (Van Breusegem et al. 1999). In ad di tion, over pro duc tion of SODs in plant chloroplasts hasbeen found to in crease pro tec tion against her bi -cides (Van Camp et al. 1996, Iannelli et al. 1999).SOD and APX are re spon si ble, re spec tively, forH2O2 pro duc tion and its scav eng ing (Yamaguchi et al. 1995, Jimenez et al. 1997). There fore, the de -creased ac tiv ity of SOD in re sponse to treat mentwith fluometuron and atrazine point out that thehigh H2O2 lev els might not be a re sult of an ac tivepro duc tion but prob a bly due to a de creased ca pac -ity of its scav eng ing. How ever, there was a re trac -tion in H2O2 con tent in both spe cies treated withrimsulfuron af ter an in cip i ent rise. Sim i lar re trac -

435

OX I DA TIVE STRESS OF HER BI CIDE-TREATED PLANTS

tions were also ob served in atrazine-treated maizeprob a bly due to the ef fi cient deg ra da tion of the her -bi cide by GSH and GST mech a nism in maize rel a -tive to broad bean (Farago et al. 1993, Nemat Alla1995, 2000, Nemat Alla and Hassan 1996, Hassan2000). In spite of the high SOD ac tiv ity in both spe -cies treated with rimsulfuron and in maize treatedwith atrazine, H2O2 con tents re mained lower thanin fluometuron-treated sam ples. These ob ser va -tions could in di cate that a de crease in per ox ide de -tox i fi ca tion was set in by fluometuron. On the other hand, the de creased ca pac ity to de com pose H2O2may be the result of an unsuccessful activation ofCAT and peroxidase enzymes.

As shown in Fig ure 5, fluometuron treat ment re -sulted in a sig nif i cant in hi bi tion in CAT ac tiv ity ofboth spe cies through out the en tire ex per i ment. Thein hi bi tion of CAT ac tiv ity was greater in broadbean than in maize. Atrazine also in duced a sig nif i -cant in hi bi tion in CAT ac tiv ity in broad bean dur ing the whole ex per i ment. In maize, the ef fect was ob -served up to the 8th day of treat ment then it be camenon sig nif i cant. Rimsulfuron, on the con trary, sig -nif i cantly stim u lated the CAT ac tiv ity up to the 12th

day in broad bean and up to the 4th day of treat mentin maize; the en zyme ac tiv ity be came there af tercom pa ra ble to con trol lev els.

There was a de crease in CAT con com i tant with anen hanced pro duc tion of car bonyl groups prob a blybe cause CAT is known to be a tar get of peroxisomal pro te ase ac tiv ity (Distefano et al. 1999). Thus thede creases in CAT ac tiv ity in the pres ent study byfluometuron and atrazine could ex plain the ac cu -mu lated lev els of H2O2. Be cause APX and GPXare ef fi cient in pre vent ing the ac cu mu la tion ofH2O2, their ac tiv i ties were checked (Fig. 6). Bothen zymes re sponded sim i larly to each her bi cide.Fluometuron re sulted in a sig nif i cant de crease inAPX and GPX ac tiv i ties in both spe cies dur ing thewhole ex per i ment; the ef fect was greater in broadbean than maize. In re sponse to atrazine, ac tiv i tiesof APX and GPX were in hib ited ei ther in broadbean dur ing the en tire ex per i ment or in maize dur -ing the first 4 and 12 days of treat ment, re spec -tively. On the con trary, rimsul furon in duced sig nif -i cant in creases in APX ac tiv ity of both spe cies up to the 8th day of treat ment and in GPX up to the 12th

day. Thereafter, activities of both enzymesdecreased to control values.

Un der these con di tions, where CAT, APX andGPX are di min ished, the cell is not fully com pe tentto re move H2O2 which would ac cu mu late to toxiclev els. More over, the de crease in SOD ac tiv itywould de lay the elim i na tion of superoxide rad i calswhereof would de crease plant tol er ance to her bi -cides. In this con text, Iannelli et al. (1999) foundthat an in creased ac tiv ity of SOD and APX is as so -ci ated to the in creased tol er ance of maize to para -quat. In ad di tion, Pyon et al. (2004) re ported thatthe ac tiv i ties of SOD, CAT and peroxidases werehigher in paraquat-re sis tant Erigeron canadensisthan in sus cep ti ble biotype. There fore, the in hi bi -tion in CAT, APX and GPX ac tiv i ties might con -firm a de fi ciency in H2O2 detoxification with aconsequent rise in its contents.

In con clu sion, treat ment of both spe cies withfluometuron de clined ac tiv i ties of SOD, CAT, APX and GPX, PSII ac tiv ity, chlo ro phyll con tents andRubisco ac tiv ity. On the other hand, there were in -creases in H2O2 con tents, lipid per ox ides and car -bonyl groups. Sim i lar re sponses were ob served inbroad bean treated with atrazine. How ever, rimsul -furon treat ment to both spe cies in duced in cip i entin creases in H2O2, lipid per ox ides, car bonylgroups, and de creases in PSII ac tiv ity, chlo ro phyllcon tents and Rubisco ac tiv ity ac com pa nied within creases in ac tiv i ties of SOD, CAT, APX andGPX. There af ter, a re trac tion in all tested pa ram e -ters to reach con trol lev els was mostly set in in di -cat ing that rimsulfuron seemed in sig nif i cant uponox i da tive stress in duc tion. The fast re cov ery in ox i -da tive stress in both spe cies treated withrimsulfuron and, to some ex tent, maize treated with atrazine might be ow ing to an in crease in her bi cidede tox i fi ca tion. In con tra dic tion, more per sis tentfluometuron in both spe cies and also atrazine inbroad bean would re sult in con stantly oc cur renceof ox i da tive stress. These find ings might in di catethat fluometuron is in volved in the gen er a tion ofox i da tive stress in both species but atrazine only inbroad bean while rimsulfuron is not in both species.

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Ref er ences

Allakhverdiev SI, Sakamoto A, Nishiyama Y,Murata N 2000. In ac ti va tion of photosystem I and II inre sponse to os motic stress in Synechococcus. Con tri bu -tion of wa ter chan nels. Plant Physiol. 122: 1201-1208.

Beyer WF, Fridovich Y 1987. As say ing for superoxidedismutase ac tiv ity: some large con se quences of mi norchanges in con di tions. Anal. Biochem. 161: 559-566.

Bowler C, Van Camp W, Van Montagu M, Inze D1994. Superoxide dismutase in plants. CRC Crit i cal Re -views in Plant Sci ences, 13: 199-218.

Brad ford MM 1976. A rapid and sen si tive method forthe quantitation of mi cro gram quan ti ties of pro tein uti -liz ing the prin ci ple of pro tein-dye bind ing. Anal.Biochem. 72: 248-154.

Briviba K, Klotz LO, Sies H 1997. Toxic and sig nal ingef fects of pho to chem i cally or chem i cally gen er ated sin -glet ox y gen in bi o log i cal sys tems. Biol. Chem. 378:1259–1265.

Buege JA, Aust SD 1972. Microsomal lipid peroxi -dation. Meth ods in Enzymology 52: 302-310.

del Rio LA, Ortega MG, Lopez AL, Gorge JL 1977. A more sen si tive mod i fi ca tion of the catalase as say withthe Clark ox y gen elec trode: ap pli ca tion to the ki neticstudy of the pea leaf en zyme. Anal. Biochem. 80:409-415.

Distefano S, Palma JM, Mc Car thy I, del Rio LA1999. Proteolytic cleav age of plant pro teins by peroxi -somal endoproteases from se nes cent pea leaves. Planta209: 308-313.

Dixit V, Pandey V, Shyam R 2001. Dif fer en tialantioxidative re sponses to cad mium in roots and leavesof pea (Pisum sativum L. cv. Azad). J. Exp. Bot. 52:1101-1109.

Dodge AD 1994. Her bi cide ac tion and ef fects on de tox i -fi ca tion pro cesses, in: C.H. Foyer, P.M. Mullineaux(Eds.), Causes of Photooxidative Stress and Ame lio ra -tion of De fense Sys tems in Plants, CRC Press, BocaRaton, pp. 219–236.

El-Shahaby OA, Nemat Alla MM, Younis ME,El-Bastawisy ZM 2002. Ef fect of kinetin on photo -synthetic ac tiv ity and car bo hy drate con tent in wa ter -logged or sea wa ter treated Vigna sinensis and Zea maysplants. Plant Biosys. 136: 277-290.

Farago S, Kreuz K, Brunold C 1993. De creasedglutathione lev els en hance the sus cep ti bil ity of maizeseed lings to metolachlor. Pestic. Biochem. Physiol. 47:199-205.

Foyer CH, Descourvieres P, Kunert KJ 1994. Pro tec -tion against ox y gen rad i cals: an im por tant de fencemech a nism stud ied in trans gen ic plants. Plant Cell En vi -ron. 17: 507-523.

Gulner G and Dodge AD 2000. Ef fect of sin glet ox y -gen gen er at ing sub stances on the ascor bic acid andglutathione con tent in pea leaves. Plant Sci. 154:127-133.

Halliwell B, Gutteridge JMC 1989. Free Rad i cals inBi ol ogy and Med i cine, 2nd edn. Ox ford Uni ver sityPress.

Hassan NM 2000. In flu ence of atrazine and fluome -turon on photosynthetic en zymes and car bo hy drate for -ma tion in Zea mays. Proc. 1st Int. Conf. Biol. Sci. Tanta1: 80-94.

Hernández JA, Jiménez A, Mullineaux P, Sevilla F2000. Tol er ance of pea (Pisum sativum L.) to long-termsalt stress is as so ci ated with in duc tion of anti-ox i dantdefences. Plant Cell En vi ron. 23: 853-862.

Hippeli S, Elstner EF 1996. Mech a nisms of ox y gen ac -ti va tion dur ing plant stress: bio chem i cal ef fects of airpol lut ants. J. Plant Physiol. 148: 249-257.

Iannelli AM, Van Breusegem F, Van Montagu M,Inze D, Massacci A 1999. Tol er ance to low tem per a tureand paraquat-me di ated ox i da tive stress in two maize ge -no types. J. Exp. Bot. 50: 523-532.

Jiménez A, Hernández JA, del Rio LA, Sevilla F1997. Ev i dence for the pres ence of the ascorba te --glutathione cy cle in mi to chon dria and peroxisomes ofpea leaves. Plant Physiol. 114: 275-284.

Keys AJ, Parry MA 1990. Ribulose bisphosphatecarboxylase/oxygenase and car bonic anhydrase. In:Meth ods in plant bio chem is try, Vol. 3, En zymes of pri -mary me tab o lism, P.M. Dey, J.B. Harborne (Eds.), Ac a -demic Press, New York, To kyo, Lon don.

Kirkwood PC 1987. Her bi cides and plant growth reg u -la tors. In: DH Hutson, TR Rob erts eds, Her bi cides, vol6, John Wiley & Sons Ltd, Chichester, New York, pp.1-55.

Kuzniak E 2002. Trans gen ic plants: an in sight into ox i -da tive stress tol er ance mech a nisms. Acta Physiol. Plant.24: 97-113.

Le vine RL, Wil liams JA, Stadtman ER, Shacter E1991. Car bonyl as says for de ter mi na tion of ox i da tivelymod i fied pro teins. Meth ods in Enzymology 233:346-363.

Martinetti L, Scarponi L, Nemat Alla MM 1995. Ef -fect of rimsulfuron and its ma jor prod uct on ALS ac tiv ity and on pro tein and starch for ma tion in maize. Brigh tonConf -Weeds, UK, 405-410.

Metzner H, Rau H, Senger H 1965. Untersuchungenzur sunchronisier barkeit einzelner-Pig ment-Man gelMutanten Von Chlorella. Planta 65: 186-199.

Nemat Alla MM 1995. Glutathione reg u la tion ofglutathione S-transferase and peroxidase ac tiv ity in her -bi cide-treated Zea mays. Plant Physiol. Biochem. 33:185-192.

437

OX I DA TIVE STRESS OF HER BI CIDE-TREATED PLANTS

Nemat Alla MM 2000. The in flu ence of naphthalic an -hy dride and 1-aminobenzotriazole on maize re sis tanceto her bi cides: A pos si ble role for glutathione S-trans -ferase in her bi cide per sis tence and de tox i fi ca tion. Agric. Med. 130: 18-26.

Nemat Alla MM, Hassan NM 1996. Rimsulfuron in ter -fer ence with syn the sis and struc tural in teg rity ofacetohydroxyacid synthase does not con trib ute to re sis -tance in Glycine max. Plant Physiol. Biochem. 34:119-126.

Noctor G, Foyer CH 1998. Ascorbate and glutathionekeep ing ac tive ox y gen un der con trol. Ann. Rev. PlantPhysiol. Plant Mol. Biol. 49: 249-279.

Okuda T, Masuda Y, Yamanaka A, Sagisaka S 1991.Abrupt in crease in the level of hy dro gen per ox ide inleaves of win ter wheat is caused by cold treat ment. PlantPhysiol. 97: 1265-1267.

Pasqualini S, Batini P, Ederli L, Porceddu A, Piccioni C, De Marchis F, Antonielli M 2001. Ef fects ofshort-term ozone fu mi ga tion on to bacco plants: re -sponses of the scav eng ing sys tem and ex pres sion of theglutathione reductase. Plant Cell En vi ron. 24: 245-252.

Pyon JY, Piao RZ, Roh SW, Shin SY, Kwak SS 2004.Dif fer en tial lev els of an ti ox i dants in paraquat-re sis tantand –sus cep ti ble Erigeron Canadensis bio types in Ko -rea. Weed Biol. Manag. 4: 75-80.

Ranieri A, Castagna A, Lorenzini G, Soldatini GF1997. Changes in thylakoid pro tein pat terns and an ti ox i -dant lev els in two wheat cultivars with dif fer ent sen si tiv -ity to sul phur di ox ide. En vi ron. Exp. Bot. 37: 125-135.

Ranieri A, D’Urso G, Nali G, Lorenzini G, SoldatiniGF 1996. Ozone stim u lates apoplastic sys tems in pump -kin leaves. Physiol. Plant. 97: 381-387.

Reinheckel T, Noack H, Lorenz S, Wiswedel I,Augustin W 1998. Com par i son of pro tein ox i da tion and al de hyde for ma tion dur ing ox i da tive stress in iso latedmi to chon dria. Free Rad i cal Res. 29: 297-305.

Sandalio LM, Dalurzo HC, Gomez M, Romero --Puetas MC, del Rio LA 2001. Cad mium-in duced

changes in the growth and ox i da tive me tab o lism of peaplants. J. Exp. Bot. 52: 2115-2126.

Snedecor W, Cochran G 1980. Sta tis ti cal Meth ods, 7th ed, The Iowa State Uni ver sity Press: Ames, IA.

Spreitzer RL 1993. Ge netic dis sec tion of rubisco struc -ture and func tion. Ann. Rev. Plant Physiol. Plant Mol.Biol. 44: 411-434.

Trebst A 1972. Mea sure ment of Hill re ac tion andphotoreduction, in: S.A. Pietro (Ed.), Meth ods in Enzy -mology, Ac a demic Press, New York, pp.146-165.

Van Breusegem F, Slooten L, Stassart J-M,Botterman J, Moens T, Montagu MV, Inze D 1999.Ef fect of over pro duc tion of to bacco MnSOD in maizechloroplasts on fo liar tol er ance to cold and ox i da tivestress. J. Exp. Bot. 50: 71-78.

Van Camp W, Capiua K, Van Montagu M, Inze D,Slooten L 1996. En hance ment of ox i da tive stress tol er -ance in trans gen ic to bacco plants over pro duc ingFe-superoxide dismutase in chloroplasts. Plan Physiol.112: 1703-1714.

Van Camp W, Van Montagu M, Inze D 1994.Superoxide dismutase. In: CH Foyer, PM Mullineaux,eds, Causes of Photooxidative Stress and Ame lio ra tionof De fense Sys tems in Plants. Boca Raton, Florida, CRC Press, pp. 317-341.

Vecchia FD, Barbato R, La Rocca N, Moro I, RascioN 2001. Re sponses to bleach ing her bi cides by leafchloroplasts of maize plants grown at dif fer ent tem per a -tures. J. Exp. Bot. 52: 811-820.

Wakabayashi K, Boger P 2004. Phytotoxic sites of ac -tion for mo lec u lar de sign of mod ern her bi cides (Part 1):The photosynthetic elec tron trans port sys tem. WeedBiol. Manag. 4: 8-18.

Yamaguchi K, Mori H, Nishimura M 1995. A novelisozyme of ascorbate peroxidase lo cal ized in glyoxy -somal and leaf peroxisomal mem branes in pump kin.Plant Cell Physiol. 36: 1157-1162.

Re ceived June 27, 2004; ac cepted April 04, 2005ed ited by G. Bartosz

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