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Differential influence of herbicide treatments on activity and kinetic parameters of C 4 photosynthetic enzymes from Zea mays

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Differential influence of herbicide treatments on activity and kinetic parameters of C 4 photosynthetic enzymes from Zea mays
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  Differential influence of herbicide treatments on activity andkinetic parameters of C 4  photosynthetic enzymes from  Zea mays Mamdouh M. Nemat Alla  * , Nemat M. Hassan, Zeinab M. El-Bastawisy Botany Department, Faculty of Science at Damietta, Mansoura University, Damietta, Egypt Received 28 March 2007; accepted 15 June 2007Available online 22 June 2007 Abstract The recommended field dose of rimsulfuron, imazethapyr, alachlor, atrazine or fluometuron differentially reduced shoot fresh and dryweight of 10-day-old maize seedlings as well as leaf protein content during the following 12 days. These reductions seemed consistentduring the whole period with fluometuron, atrazine and alachlor but appeared to be nullified by the 5th day of treatment with rimsul-furon and imazethapyr. On the other hand, all herbicides mostly provoked significant inhibitions in specific activities of phospho enol  pyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.82), pyruvate phosphate dikinase(PPDK, EC 2.7.9.1) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in leaves during the first 2 days. There-after, the inhibition was recovered in samples treated with rimsulfuron and imazethapyr, leveled off with alachlor but consistently aug-mented with atrazine and fluometuron. The kinetic characterization showed that rimsulfuron or imazethapyr unchanged  V  max  of allenzymes in vitro, however,  V  max  of PEPC, PPDK and Rubisco were decreased in vivo. Nevertheless, atrazine or fluometuron substan-tially reduced  V  max  of all enzymes while alachlor showed a reduction in this value of PEPC, MDH and Rubisco. Thus atrazine, fluo-meturon and, to a lower extent, alachlor reduced concentrations of all enzymes as well as rimsulfuron and imazethapyr for onlyRubisco. On the contrary,  K  m  values of all enzymes were progressively increased by all herbicides indicating that the different herbicidesaltered the structural integrity of all enzymes. These findings conclude that rimsulfuron or imazethapyr competitively inhibited MDH butrevealed mixed inhibition to PEPC, PPDK and Rubisco. Atrazine or fluometuron revealed mixed inhibitions to all enzymes whereasalachlor seemed to be either a competitive inhibitor to PPDK or a mixed inhibitor to PEPC, MDH and Rubisco.   2007 Elsevier Inc. All rights reserved. Keywords:  Herbicides; Kinetic parameters; Malate dehydrogenase; Phospho enol  pyruvate carboxylase; Pyruvate phosphate dikinase; Rubisco;  Zea mays 1. Introduction Sulfonylurea herbicides such as rimsulfuron [1-(4,6-dimethoxypyrimidin-2-y1)-3-(pyridylsulfonyl)urea] andimidazolinones like imazethapyr [( RS  )-5-ethyl-2-[4-isopro-pyl-4-methyl-5-oxo-2-imidazolin-2-yl]nicotinic acid] inhibitacetohydroxyacid synthase [AHAS, EC 4.1.3.18], the firstenzyme in valine, leucine and isoleucine biosynthesis [1,2].On the other hand, alachlor [2-chloro-2,6-diethyl- N  -meth-oxymethylacetamlide], the  a -chloroacetanilide herbicide,affectssomemetabolicprocessesduringinitialseedgermina-tion and further development such as protein synthesis [3].Atrazine [2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine] and fluometuron [l,l-dimethy1-3-( a , a , a -trifluoro- m -tolyl)urea], the  S  -triazine and phenylurea herbicides,respectively, block the electron transport chain with a con-sequent inhibition of ATP and NADPH formation leadingto photooxidative destruction of pigments [4,5]. In spite of the varied modes of herbicide action, photosyntheticenzymes are directly or indirectly influenced in treatedplants.TheC 4 photosyntheticpathwayisconsideredtohavethe highest efficiency and potential productivity of all formsofhigherplantphotosynthesis[6].OfthemostimportantC 4 photosynthetic enzymes are PEPC, NADP-MDH andPPDK. These enzymes have been shown to exert metabolic 0048-3575/$ - see front matter    2007 Elsevier Inc. All rights reserved.doi:10.1016/j.pestbp.2007.06.005 * Corresponding author. Fax: +20 57 2403868. E-mail address:  mamnematalla@mans.edu.eg (M.M. Nemat Alla). www.elsevier.com/locate/ypest Pesticide Biochemistry and Physiology 89 (2007) 198–205 PESTICIDE Biochemistry & Physiology  control over light-saturated C 4  photosynthesis [7]. PPDKcatalyses the conversion of pyruvate to PEP, the primaryCO 2  acceptor which is carboxylated by PEPC to oxaloace-tate (OAA), the substrate of MDH [8]. Moreover, Rubiscois the key enzyme in photosynthetic carbon fixation andthe most abundant protein in plants [9,10]. It catalyses theirreversible carboxylation of ribulose-1,5-bisphosphate toform two 3-phosphoglyceric acid molecules. However, therate of the reaction is extremely slow, and Rubisco mustbe activated to become catalytically competent. ActivationisachievedbyRubiscoactivasewhichcanremoveinhibitorsfrom Rubisco’s catalytic sites [11]. Photosynthetic efficiencyand biomass accumulation are highly correlated with Rubi-sco and PPDK [12]. These enzymes might be affected byboth protein- and photosynthesis-inhibiting herbicidesprobably because of a decrease in enzyme concentrationand/or a retraction in enzyme–substrate affinity [13]. Thekinetic parameters define the rate behaviour of an enzyme-catalysed reaction. This study was aimed to examine theactivities and kinetics parameters of C 4  photosyntheticenzymes from maize treated with the recommended fielddoseofherbicideswithvariedmodesofaction(rimsulfuron,imazethapyr, alachlor, atrazine and fluometuron). 2. Materials and methods  2.1. Plant material and growth conditions Grains of maize ( Zea mays  L., Giza 2) were surface ster-ilized by immersing in 3% sodium hypochlorite solution for10 min, thoroughly washed, soaked for 8 h and germinatedin sand/clay soil (1/1, v/v) in plastic pots (25 cm diame-ter  ·  20 cm height). The pots were kept under controlledconditions (14 h photoperiod with 450–500  l mol m  2 s  1 photosynthetic photon flux density, 75–80% relativehumidity and 28/14   C day/night regime). After 10 days,seedlings were thinned to 4 per pot and pots were dividedinto six groups; one to serve as a control and one for eachherbicide treatment at a dosage corresponding to the rec-ommended field dose (0.015, 0.035, 3.36, 1.79 and2.98 kg ai ha  1 for rimsulfuron, imazethapyr, alachlor,atrazine and fluometuron, respectively). The quantity of each herbicide was solubilized in a suitable amount of water enough to spray the surface area of each pot inone direction and crosswise. The herbicides were appliedonly once. At harvest, after the seedlings have beenexposed to light for at least 4 h, leaves were collected atzero time (just before herbicide application) and also after1, 2, 5 and 12 days from treatment, washed with copiousamounts of water and blotted dry with paper towels.  2.2. Determination of protein content Extraction of protein was carried out in 80 mM Tris– HCl, pH 7.4 [14]. After centrifugation at 14,000  g   for5 min, the extracted protein was precipitated overnight at4   C by adding chilled (10%, w/v) trichloroacetic acid inacetone. Protein pellets were separated by centrifugationat 12,000  g   for 15 min and reconstituted in the buffer. Pro-tein was determined using Coomassie Brilliant Blue G-250at 595 nm [15].  2.3. Extraction and assay of PEPC, MDH and PPDK  Extraction and assay procedures of PEPC, NADP-MDH and PPDK were performed as described by Ashtonet al. [16]. About 2 g of pre-illuminated leaves were homog-enized at 4   C with the extraction buffer containing 50 mMHepes–KOH (pH 7.5) and 10 mM dithiothreitol (DTT).For extraction of PEPC only, 5 mg ml  1 bovine serumalbumin, 5 mM MgCl 2  and 2 mM KPO 4  were added. Themixtures were centrifuged at 10,000  g   at 48  o C for 10 min.The supernatants were applied on Sephadex G-25 (Phar-macia) equilibrated with Hepes–KOH buffer.The activity of PEPC was assayed at 340 nm by follow-ing the reduction of OAA by NADH in the presence of MDH. The reaction mixture contained 25 mM Tris–HCl(pH 8.0), 5 mM MgCl 2 , 2 mM DTT, 1 mM NaHCO 3 ,5 mM glucose-6-phosphate, 5 mM PEP, 0.2 mM NADHand 2 U of MDH. The activity of MDH was measured inthe direction of OAA reduction by following the oxidationof NADPH at 340 nm. Reaction mixture contained 25 mMTricine–KOH (pH 8.3), 70 mM KCl, 1 mM EDTA, 1 mMDTT, 1 mM OAA and 0.2 mM NADPH. The activityassay of PPDK was performed at 340 nm in the forwarddirection by coupling the production of PEP to NADHvia PEPC and MDH. The reaction mixture contained25 mM Hepes–KOH (pH 8.0), 8 mM MgSO 4 , 10 mMDTT, 10 mM NaHCO 3 , 2 mM pyruvate, 5 mM(NH 4 ) 2 SO 4 , 1 mM glucose-6-phosphate, 1 mM ATP,2.5 mM KPO 4 , 0.2 mM NADH, 0.5 U PEPC and 2 U of MDH.  2.4. Extraction and assay of Rubisco Extraction and assay procedures were performedaccording to Keys and Parry [17]. Extraction buffer con-tained 20 mM Tris–HCl (pH 8.0), 10 mM NaHCO 3 ,10 mM MgCl 2 , 1 mM EDTA, 5 mM DTT, 0.002% chlo-rohexidine diacetate (Hibitane) and 1% (w/v) polyvinylpyr-rolidone. The homogenate was centrifuged at 5000  g   for15 min and the supernatant was treated with solid(NH 4 ) 2 SO 4  to give 35% saturation, centrifuged at 20,000  g  for 15 min and the supernatant was raised to 55% satura-tion. After centrifugation at 20,000  g   for 15 min, the pelletwas dissolved in 20 mM Tris–HCl (pH 8.0) containing1 mM DTT, 1 mM MgCl 2  and 0.002% Hibitane.Rubisco activity was assayed in 50 mM 4-(2-hydroxy-methyl)1-piperazine ethane sulfonic acid (Hepes, pH 7.8),containing 10 mM NaHCO 3 , 20 mM MgCl 2 , 0.66 mMribulose-1,5-bisphosphate (RuBP), 0.2 mM NADPH,5 mM ATP, 5 mM creatine phosphate, 2.0 U creatinephosphokinase, 2.8 U glyceraldehyde-3-phosphate dehy-drogenase and 2.0 U phosphoglycerate kinase. The con- M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 89 (2007) 198–205  199  tents were allowed to react at 30   C, during which thedecrease in absorbance at 340 nm was measured.  2.5. Determination of kinetic parameters Two sets of enzyme extracts were used for determinationof   K  m  and  V  max . The first set was obtained from leaves of 10-day-old untreated control samples collected just beforeherbicide application (at zero time). The reaction mixtureof each enzyme contained rimsulfuron, imazethapyr, ala-chlor, atrazine or fluometuron in a final concentration of 0.075  l M, 0.183  l M, 125  l M, 72  l M or 114  l M, respec-tively. The other set was obtained from leaves of treatedseedlings 5 days following treatments but without anyadded herbicides to the reaction mixtures. The activitiesof PEPC, MDH, PPDK and Rubisco were determined atvaried substrate concentrations ([ S  ]). The substrate concen-tration of PEPC ([PEP]), MDH ([OAA]), PPDK ([pyru-vate]) and Rubisco ([RuBP]) varied from 1 to 20 mM, 0.2to 4 mM, 0.4 to 10 mM and 0.2 to 2 mM, respectively. Aplot of velocity ( v ) against [ S  ] gives a curve belongs toMichaelis–Menten plot [13]. The values of   V  max  and  K  m were obtained from Hanes equation: [ S  ]/ V   = [ S  ](1/ V  max ) +  K  m / V  max , the linear transformation of Michaelis– Menten equation:  v  = ( V  max [ S  ])/( K  m  + [ S  ]). A plot of [ S  ]/ v  against [ S  ] should give a straight line with an ordinateintercept of   K  m / V  max , a slope of 1/ V  max  and an abscissaintercept of   K  m  (Hanes plot).Each experiment was repeated twice in triplicate, so thatthe mean obtained was for six replicates. All data were sta-tistically analysed using the least significant differences(LSD) method [18]. 3. Results and discussion 3.1. Changes in growth and protein content Treatments of maize with the recommended field dose of rimsulfuron, imazethapyr, alachlor, atrazine and fluometu-ron significantly reduced the magnitudes of increase inshoot fresh and dry weight of 10-day-old maize seedlingsduring the following 12 days (Fig. 1). This trend consis-tently augmented by fluometuron, atrazine and alachlorduring the whole period but appeared to be nullified bythe 5th day of treatment with rimsulfuron and imazetha-pyr. On the other hand, all herbicides resulted in significantdecreases in leaf protein content. The reduction continuedup to the 12th day in seedlings treated with atrazine andfluometuron. However, there was a recovery detected fromthe 5th day following treatment with rimsulfuron, imazeth-apyr and alachlor.Thus all herbicides induced a general reduction in maizegrowth and protein content; however, these reductions dif-fered for the different herbicides. The effects were beingleast with rimsulfuron and imazethapyr. Nevertheless, atra-zine and fluometuron exerted the most reductive effectswhereas the effect of alachlor seemed moderate. Severalreports indicated reduction in growth of several plant spe-cies after the application of these herbicide groups [19–22].The reduction in plant growth could result from varied dis-turbances in certain processes by the herbicides, e.g., thoserelated to nitrogen metabolism and photosynthesis [19,23– 25]. Really, sulfonylureas and imidazolenones causegrowth cessation of several plant species by interferingbranched-chain amino acids biosynthesis [1,2,23,24]. Onthe other hand,  a -chloroacetanilides affect protein synthesisand seed germination [3,21,26,27] while  S  -triazines andphenylureas interfere with electron transport chain[4,5,19,21]. Therefore, it could be predicted that proteinsynthesis might be affected by these herbicides eitherdirectly or indirectly with a consequence alteration in pro-tein available for enzyme structure and function amongwhich the C 4  photosynthetic enzymes. 3.2. Changes in enzyme activities As shown in Fig. 2, the specific activities of PEP, MDH,PPDK and Rubisco in maize leaves were significantly 030060090012000 12    S   h  o  o   t   f  r  e  s   h  w  e   i  g   h   t   (  m  g  p   l  a  n   t   -   1    ) Days after treatment03060900 12    S   h  o  o   t   d  r  y  w  e   i  g   h   t   (  m  g  p   l  a  n   t   -   1    ) Days after treatment024680 12    P  r  o   t  e   i  n  c  o  n   t  e  n   t   (  m  g  g   -   1     f  r  e  s   h  w  e   i  g   h   t   ) Days after treatment4 8 4 8 4 8 Fig. 1. Influence of herbicides on (a) shoot fresh weight; (b) shoot dry weight and (c) leaf protein content of 10-day-old maize during the following 12 days. s , Control; d , rimsulfuron; h , imazethapyr; j , alachlor;  D , atrazine; m , fluometuron. Each value is the mean of six replicates. Vertical bars representLSD at 5% level.200  M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 89 (2007) 198–205  decreased during the first 2 days from treatment with therecommended field dose of the applied herbicides. Thereaf-ter, complete recoveries seemed to set in only in samplestreated with rimsulfuron or imazethapyr. In contrast, theinhibitions in the activities of all enzymes were augmentedby atrazine or fluometuron. However, the effect of alachlorwas overcome either on the 12th day for PEPC and PPDKor by the 5th day for Rubisco.Several herbicides inhibit the activities of many enzymesinvolved in the primary metabolism. Young leaves are gen-erally affected because these organs act as sinks of herbi-cides [28]. The activities of PEPC and Rubisco in maizewere significantly inhibited by rimsulfuron [23]. Moreover,inhibitions in the activities of AHAS, glutamine synthetase(EC 6.3.1.2.) and glutamate synthase (EC 1.4.1.14) werealso reported. A similar significant inhibition in the activityof Rubisco was shown in soybean following imazethapyrtreatment [29]. Propachlor, chlorimuron-ethyl or imazetha-pyr resulted in inhibitions in the activities of Rubisco andMDH in  Vicia faba  [27]. The differential inhibition inenzyme activities by the varied herbicides in the presentstudy might be attributable to the use of differential doses,the strength of the target site and/or the rates of detoxifica-tion in the plant tissues. Moreover, a decrease in proteinavailable for an enzyme and/or in substrate concentrationwithin plant tissues would lower product formation andsubsequently decrease the enzyme activity. Thus, a dropin PPDK activity would insufficiently yield PEP for PEPCaction. Under these circumstances, the low levels of PEPavailable for PEPC would result in low formation of OAA for MDH function with slight production of CO 2 in bundle sheath for Rubisco action. The observed inhibi-tion in these activities might be a consequence of a lossof   de novo  synthesis of the enzyme proteins. Indeed signif-icant reductions in protein synthesis were observed in sev-eral plant species following treatments with manyherbicides [24,29–32]. Indeed, Parry et al. [33] indicated that the amount of leaf enzymes is controlled by their ratesof synthesis and degradation. On the other hand, severeconditions do decreases in protein content and activitiesof many enzymes [34–37]. As a result of interference withbranched-chain amino acids or protein synthesis, rimsulfu-ron and imazethapyr or alachlor would directly inhibit pro-tein formation available for enzyme structure. However,fluometuron and atrazine which inhibit photosynthesiswould indirectly retard protein skeleton formation andsubsequently enzyme activity. 3.3. Changes in kinetic parameters So, the inhibition of enzyme activities by herbicidescould result from a decrease in its concentration in additionto the interference with the structural integrity, which canbe related to isoenzyme distribution and/or enzyme–sub-strate affinity. Therefore, Michaelis–Menten plot showactivities of the extractable PEPC, MDH, PPDK andRubisco from the untreated maize leaves at the applicationtime as a function of their substrates without or with thepresence of herbicides in vitro (Fig. 3a–d). These assayswere performed also for enzymes extracted from leaves of treated seedlings 5 days following treatment but withoutherbicides added to the reaction mixtures (Fig. 3e–h).The velocities were generally lower for enzymes extractedfrom treated than untreated tissues. Rimsulfuron, imazeth-apyr and to some extent alachlor slightly, if any, loweredthe velocities of all enzymes. However, atrazine or fluo-meturon provoked greater drop in the enzyme velocitiesthan the other herbicides did.The differential inhibition in the specific activities of these enzymes might result from changes in the kineticparameters,  K  m  and  V  max . These parameters are useful asdescriptors of the enzymes. They define the rate behaviourof an enzyme-catalysed reaction as substrate concentrationis varied. The results tend to indicate a decrease in  V  max  of  00.20.40.60.80 12    P   P   D   K  s  p  e  c   i   f   i  c  a  c   t   i  v   i   t  y   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1   p  r  o   t  e   i  n   h   -   1    ) Days after treatment00.20.40.60.80 12    M   D   H  s  p  e  c   i   f   i  c  a  c   t   i  v   i   t  y   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) Days after treatment00.20.40.60.80 12    P   E   P  -   C  s  p  e  c   i   f   i  c  a  c   t   i  v   i   t  y   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) Days after treatment 00.20.40.6 0 12Days after treatment    R  u   b   i  s  c  o  s  p  e  c   i   f   i  c  a  c   t   i  v   i   t  y   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) 4 8 4 8 4 8 4 8 Fig. 2. Influence of herbicides on specific activity of (a) phospho enol  pyruvate carboxylase (PEPC); (b) malate dehydrogenase (NADP-MDH); (c) pyruvatephosphate dikinase (PPDK) and (d) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in leaves of 10-day-old maize during the following 12days.  s , Control;  d , rimsulfuron;  h , imazethapyr;  j , alachlor;  D , atrazine;  m , fluometuron. Values are expressed as decrease in absorbancemg  1 protein min  1 . Each value is the mean of six replicates. Vertical bars represent LSD at 5% level. M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 89 (2007) 198–205  201  all enzymes by all herbicides accompanied with a remark-able rise in  K  m . This trend seems more obvious for enzymesextracted from treated than untreated seedlings. Since  V  max is a function of the enzyme concentration, a decrease in itsvalue consequently suggests a decrease in the enzyme activesites participate in the reaction. On the other hand,  K  m reflects an enzyme’s affinity for its substrate and can berelated to isoenzyme distribution, enzyme–substrate affin-ity, etc. [8,13]. Therefore, an increase in  K  m  values couldconfirm that herbicides interfered with the enzyme struc-tural integrity.In confirmation, Hanes plot was used to accuratelydetermine the values of   K  m  and  V  max  (Fig. 4). There wereprogressive increases in the ordinate intercept ( K  m / V  max )of all enzymes by the different herbicides. Thereupon, itis possible to deduce that herbicides might increase  K  m and/or decrease  V  max  values. The slope values of lines (1/ V  max ) were differentially increased indicating that these her-bicides might reduce  V  max  values of all enzymes. Thesechanges were most pronounced with fluometuron followedby atrazine and least with rimsulfuron and imazethapyrwith an intermediary influence of alachlor. As a whole, V  max  values of enzymes extracted from the treated seed-lings were lower than those of the untreated seedlings.For easier interpretation of results, values of kineticparameters are determined and obtained by data interpola-tion from equations presented in Fig. 4 (Table 1).  V  max  val-ues of all enzymes extracted from the untreated tissues werenon-significantly changed by the presence of rimsulfuronor imazethapyr in the reaction mixtures. Nevertheless, ala-chlor significantly decreased  V  max  of PEPC, MDH andRubisco but had non-significant effects upon PPDK. Atra-zine and fluometuron significantly and substantiallyreduced these values of all enzymes. Enzymes extractedfrom treated seedlings were lower in their  V  max  values thanblanks. All herbicides caused a drop in  V  max  of all enzymesexcept for that of MDH which seemed to be unchanged byrimsulfuron and imazethapyr. On the contrary,  K  m  valuesof all enzymes increased by the different herbicides. Themagnitude of increase was most pronounced with fluo-meturon. Anyway,  K  m  values highly increased by herbi-cides either in vitro or in vivo.In accordance,  V  max  and  K  m  were shown to be influ-enced by several herbicides [29,32,38–40]. Bukowska andHutnit [39] concluded that significant decreases of   V  max and  K  m  were observed in the activity of acetyl cholinester-ase incubated with 2,4-dichlorophenol and catechol, reveal-ing mixed type of inhibition. They reported that these 00.10.20.30.40.50   v  o   f   M   D   H   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) [oxaloacetate]00.20.40.60 10 15 20   v  o   f   P   E   P   C   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) [phospho enol pyruvate]00.20.40.60 10   v  o   f   P   P   D   K   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) [pyruvate] 00.10.20.30 0.5 1 1.5 2 [ribulose 1,5-bisphosphate]   v  o   f   R  u   b   i  s  c  o   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) 00.10.20.30.40 0.5 1 1.5 2   v  o   f   R  u   b   i  s  c  o   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) [ribulose 1,5-bisphosphate] 00.20.40.60 10 [pyruvate]   v  o   f   P   P   D   K   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) 00.20.40.60.80 10 15 20   v  o   f   P   E   P   C   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) [phospho enol pyruvate]00.20.40.60[oxaloacetate]   v  o   f   M   D   H   (   d  e  c  r  e  a  s  e   i  n  a   b  s  o  r   b  a  n  c  e  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) 5  1 2 3 4  2 4 6 8 5 1 2 3 4 2 4 6 8 Fig. 3. Michaelis–Menten plot of (a) phospho enol  pyruvate carboxylase (PEPC); (b) malate dehydrogenase (NADP-MDH); (c) pyruvate phosphatedikinase (PPDK) and (d) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) extracted from leaves of untreated 10-day-old maize as a function of substrate concentration ([ S  ]) in the presence of herbicides in vitro. (e, f, g and h) Represent plots of enzymes extracted from leaves of treated seedlings 5days following treatment. s , Control; d , rimsulfuron;  h , imazethapyr; j , alachlor;  D , atrazine; m , fluometuron.202  M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 89 (2007) 198–205
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