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Effect of metribuzin, butachlor and chlorimuron-ethyl on amino acid and protein formation in wheat and maize seedlings

Effect of metribuzin, butachlor and chlorimuron-ethyl on amino acid and protein formation in wheat and maize seedlings
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  Effect of metribuzin, butachlor and chlorimuron-ethyl on amino acidand protein formation in wheat and maize seedlings M.M. Nemat Alla  * , A.M. Badawi, N.M. Hassan, Z.M. El-Bastawisy, E.G. Badran Botany Department, Faculty of Science at Damietta, Mansoura University, Damietta, Egypt Received 21 January 2007; accepted 10 July 2007Available online 31 July 2007 Abstract Application of the recommended field dose of metribuzin, butachlor and chlorimuron-ethyl to 10-days-old wheat and maize seedlingsdifferentially reduced shoot fresh and dry weights during the following 16 days. Metribuzin was the most reductive while butachlor wasthe least. The herbicides slightly affected the activities of nitrate reductase (NR, EC and nitrite reductase (NiR, EC butgreatly inhibited glutamine synthetase (GS, EC and glutamate synthase (GOGAT, EC activities. Meanwhile, there weresignificant accumulations of ammonia and soluble-N accompanied by diminutions in total-N and protein contents; metribuzin exertedthe greatest changes. Additionally, aliphatic, aromatic and total amino acids in both species were mostly elevated by the three herbicides;however, valine, leucine and isoleucine were decreased by only chlorimuron-ethyl. These results could conclude that herbicides, partic-ularly metribuzin, cause a shortage in ammonia assimilation and subsequently a decrease in protein formation. Moreover, the elevationof soluble-N and amino acids appeared to result from breakdown of the pre-existing protein, a state that seemed consistent in seedlingstreated with metribuzin and, to some extent chlorimuron-ethyl but recovered in those treated with butachlor.   2007 Elsevier Inc. All rights reserved. Keywords:  Wheat; Maize; Herbicides; Nitrogen metabolism; Nitrogen-related enzymes 1. Introduction Herbicides drastically influence all aspects of primaryand secondary metabolism in crops when given to controlundesired weeds. Metribuzin [4-amino-6- tert -butyl-4,5-dihydro-3-methylthio-1,2,4-triazin-5-one], a triazinone her-bicide, inhibits photosynthesis [1]. It interferes with photo-synthetic electron transport between the primary andsecondary acceptor of PSII [2]. Thus CO 2  assimilation isdecreased leading to starvation and reactive oxygen speciesare formed causing oxidative stress [3,4]. Butachlor [ N  -but-oxymethyl-(2-chloro-2,6-diethylacetanilide], the chloroace-tanilide herbicide, affects seed germination, lipidmetabolism, pigment and gibberelic acid synthesis, celldivision, cell permeability, mineral uptake and disturb theabsorption and incorporation of amino acids into protein[5,6]. The sufonylurea herbicide, chlorimuron-ethyl [Ethy-2-(((((4-chloro-6-methoxypyrimidin-2-yl)amino)carbonyl)amino) sufonyl) benzoate], inhibits acetohydroxyacid syn-thase (AHAS), the key enzyme for biosynthesis of valine,leucine and isoleucine [7,8]. These herbicides have variedmodes of action, however, they could interfere with nitro-gen cycle either directly or indirectly. Ammonia is regardedas very important for plant survival; however, its accumu-lation would be harmful. Among the major processes liber-ating ammonia is the reduction of nitrate and nitrite by NRand NiR, respectively [9]. Then ammonia is assimilatedinto an organic form by a two-step reaction. In the firststep, glutamine is produced from the amination of   L -gluta-mate by GS while in the second, two molecules of   L -gluta-mate are produced from the reaction of glutamine and2-oxoglutarate by GOGAT [10,11]. So, the changes of these activities might disturb the synthesis of amino acidsand protein. Many plant species could tolerate such modi-fications. Therefore, the present work was aimed to ascer-tain the differential tolerance of wheat and maize to 0048-3575/$ - see front matter    2007 Elsevier Inc. All rights reserved.doi:10.1016/j.pestbp.2007.07.003 * Corresponding author. Fax: +20 57 2403868. E-mail address: (M.M. Nemat Alla).  Available online at Pesticide Biochemistry and Physiology 90 (2008) 8–18 PESTICIDE Biochemistry & Physiology  metribuzin, butachlor and chlorimuron-ethyl throughchecking amino acids and protein as well as activities of nitrogen-related enzymes. 2. Materials and methods  2.1. Plant materials and growth conditions Grains of wheat ( Triticum aestivum  L. Giza 168) andmaize ( Zea mays  V.S.C.129) were surface sterilized byimmersing in 3% sodium hypochlorite solution for10 min, thoroughly washed, soaked for 8 h and germinatedin quartz sand in plastic pots (25 cm diameter  ·  20 cmheight). Each pot contained five germinating seeds. Thepots were kept in a glasshouse at 22/10 or 28/14   C day/night for wheat or maize, respectively, under a 14-h photo-period at 450–500  l mol m  2 s  1 photosynthetic photonflux density, and 75–80% relative humidity. When seedlingswere 10-days-old, one-fourth strength Hoagland solutionwas used instead of water and pots were divided into fourgroups. One was left to serve as control and one for eachherbicide treatment at the recommended field dose(1.0 kg ha  1 , 3.0 L ha  1 and 20.0 g ha  1 for metribuzin,butachlor and chlorimuron-ethyl, respectively). The quan-tity of each herbicide was calculated in relation to the sur-face area per pot and solubilized in a suitable amount of water enough to spray the surface area of each pot inone direction and crosswise. Shoots of both species werecollected just before herbicide application (zero time) andalso after 4, 8, 12 and 16 days from treatments, rinsed withcopious amounts of water and dried by plotting with papertowels.  2.2. Assay of NADH–nitrate reductase [NADH–NR, EC] Extraction and assay were carried out according to Nak-agawa et al. [12]. Plant tissue (10 g) was homogenized in250 mM KH 2 PO 4 /K 2 HPO 4  (pH 8.0) containing 1 mM phe-nyl methyl sulfonyl fluoride (PMSF), 5% isopropyl alcohol,1 mM  b -mercaptoethanol, 1 mM EDTA, 5 mM KNO 3 ,0.02 mM FAD and 6.3% polyclar AT (w/v). The homoge-nate was centrifuged at 10,000  g   for 15 min. The reactionmixture contained 25 mM KH 2 PO 4 /K 2 HPO 4  (pH 7.5),5 mM KNO 3  and 0.1 mM NADH was incubated at35   C for 30 min. The diazo-coupling reagents: 1% sulpha-nilamide (w/v) in 3 M HCl and 0.02%  N  -(1-naphthyl)-eth-ylenediamine dihydrochloride (w/v) was added. After20 min, the absorbance was read at 540 nm to measurethe nitrite produced.  2.3. Assay of nitrite reductase [NiR, EC] Fresh tissue (10 g) was homogenized in 50 mM Tris– HCl buffer (pH 7.9), containing 5 mM cysteine, 2 mMEDTA, 10 mM  b -mercaptoethanol, 10% glycerol and 5%polyclar AT (w/v) [13]. The reaction mixture contained33 mM KH 2 PO 4 /K 2 HPO 4  (pH 7.5), 2 mM KNO 3 , 1 mMmethyl viologen and 11.6 mM sodium dithionite [14]. Afterincubation at 30   C for 20 min in open tubes, the reactionwas stopped by vigorous shaking. The diazo-couplingreagents: 1% sulphanilamide (w/v) in 3 M HCl and 0.02% N  -(1-naphthyl)-ethylenediamine dihydrochloride (w/v)was added. After 20 min, the absorbance was read at540 nm to measure the nitrite lost.  2.4. Assay of glutamine synthetase [GS, EC] According to Lea et al. [10], fresh weight (10 g) wasextracted in 50 mM Tris–HCl (pH 7.8) containing 1 mMsodium glutamate and 10% ethandiol (v/v). The homoge-nate was centrifuged at 15,000  g   for 15 min. Assay of GSwas performed in 50 mM glutamate, 5 mM hydroxylaminehydrochloride, 50 mM MgSO 4  and 20 mM ATP in100 mM Tris–HCl (pH 7.8). After incubation at 35   C for1 h, the reaction was terminated by ferric chloride reagent(0.67 M FeCl 3 , 0.37 M HC1 and 20% trichloroacetic acid,TCA, v/v). The absorbance was read at 540 nm to measurethe  c -glutamylhydroxamate formed.  2.5. Assay of glutamate synthase [NADH–glutamine oxo glutarate amino transferase, NADH–GOGAT, EC] Fresh weight (10 g) was extracted in 50 mM KH 2 PO 4 /K 2 HPO 4  (pH 7.5) containing 10 mM KCl, 5 mM EDTA,12.5 mM  b -mercaptoethanol, 1 mM PMSF, 2 mM 2-oxo-glutarate, 20% ethandiol (v/v) and 0.05% Triton X-100(w/v) [15]. The assay mixture contained 100 mMKH 2 PO 4 /K 2 HPO 4  (pH 7.5), 0.1 mM NADH, 10 mM glu-tamine, 10 mM 2-oxoglutarate [16]. The reaction was fol-lowed up at 340 nm for about 10 min at 30   C tomeasure the consumption of NADH.  2.6. Determination of nitrogenous constituents Ammonia and soluble-N were extracted with water [17]from dried ground tissue (3 g). Ammonia was determined,using the indophenol method, in saturated boric acid solu-tion, hypochlorite (chlorine water, saturated solution) and8% phenol solution [18]. The contents were placed in asteam bath for 3 min then removed; cooled rapidly and3 M NaOH was added. After 5 min, the absorbance wasread at 625 nm. Both soluble-N in the water extract(3 ml) and total-N in the dried tissue (50 mg) were digestedand converted into ammonia distillate using the conven-tional micro-Kjeldahl method [19] and determined usingthe indophenol method as described above.  2.7. Determination of protein content The extraction was carried out in 80 mM Tris–HCl, pH7.4 [20]. After centrifugation at 14,000  g   for 5 min, chilled10% TCA (w/v) in acetone was added to precipitateprotein over night at 4   C. Protein pellets were separated M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18  9  by centrifugation at 12,000  g   for 15 min, washed withchilled acetone, allowed to dry in air and reconstituted inthe buffer. Protein was determined using Coomassie bril-liant blue G-250 at 595 nm [21].  2.8. Determination of amino acid concentrations According to Rhodes et al. [22], amino acids wereextracted with methyl alcohol and phase separated by chlo-roform and distilled water. The aqueous phase was dried,redissolved in water and eluted on Dowex 50-H + withNH 4 OH. The eluent was dried, redissolved in the mixture(methanol:1 M sodium acetate:triethylamine, TEA, 2:2:1)and dried again. Derivatization was performed using themixture (methanol:water:TEA:phenylisothiocyanate,7:1:1:1) [23]. After dryness, 5 mM NaH 2 PO 4 /Na 2 HPO 4 (pH 7.6) containing 5% acetonitrile (v/v) were added andinjected into the HPLC. The system was adjusted as fol-lows: solution A (140 mM sodium acetate containingTEA and adjusted to pH 6.4 with glacial acetic acid), solu-tion B (acetonitrile:water, 60:40), the flow rate(0.8 ml min  1 ), the gradient flow was performed withrespect to solution B (10% at start, 6 min to 12.5%,32 min to 58%, 33 min step 100% and 12 min wash forre-equilibration at 10% again).All values are means of at least six determinations fromtwo independent experiments. The full data were statisti-cally analyzed using the least significant differences (LSD)test at 5% level [24]. 3. Results Application of the recommended field dose of metribu-zin, butachlor and chlorimuron-ethyl to 10-days-old wheatand maize seedlings resulted in differential significantdecreases in shoot fresh weight below the control values(Fig. 1). The significant reduction in both species was con-tinued by metribuzin up to the end of the experimental per-iod (16 days after treatment). However, the reduction bybutachlor or chlorimuron-ethyl seemed to be leveled off  01503004500 4 8 12 16    S   h  o  o   t   f  r  e  s   h  w  e   i  g   h   t   (  m  g  p   l  a  n   t   -   1    ) (A) 040080012000 4 8 12 16 (B) Control Metribuzin Butachlor Chlorimuron-ethyl 02040600 4 8 12 16    S   h  o  o   t   d  r  y  w  e   i  g   h   t   (  m  g  p   l  a  n   t   -   1    ) (A) Days after treatment 040801200 4 8 12 16 (B) Days after treatment Fig. 1. Changes in fresh and dry weights of (A) wheat and (B) maize shoots as a result of treatment with the recommended field dose of metribuzin,butachlor and chlorimuron-ethyl. Data are means (±SD) of at least six replications from two independent experiments. Vertical bars represent LSD at 5%level.10  M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18  after 8 days of treatment in wheat and 4 days in maize. Inthe same pattern, the herbicides markedly reduced dry mat-ter; metribuzin was much more reductive than butachlorand chlorimuron-ethyl. The effect of metribuzin extendedduring the entire experimental period whereas butachlorand chlorimuron-ethyl showed their significant reductionsonly during the first 4 days from treatment.The results depicted in Fig. 2 show that NR activity wassignificantly increased by metribuzin up to the 12th day inwheat and the 8th day in maize relative to the untreatedcontrols. However, butachlor and chlorimuron-ethylcaused significant decreases up to the 8th day in wheatand the 4th day in maize. On the contrary, there was a sig-nificant increase in NiR activity of both species by the threeherbicides during the first 4 days of the experiment. There-after, the increases, if any, became non-significant.As can be seen from Fig. 3 activities of GS and GOGATwere significantly inhibited by herbicide application, themagnitude of inhibition was most pronounced by metribu-zin. The inhibition in GS activity was detected up to the12th day from metribuzin treatment. Both butachlor andchlorimuron-ethyl resulted in a significant inhibition dur-ing the first 4 days in wheat and 8 days in maize. Similarly,GOGAT activity of both species was significantly inhibitedby metribuzin during the whole experiment. To a lesserextent, butachlor and chlorimuron-ethyl exerted significantinhibition in the enzyme activity of both species during thefirst 4 days; the inhibition was extended up to the 8th dayeither in wheat by butachlor or in maize by chlorimuron-ethyl.In Fig. 4 all herbicides induced much accumulations of ammonia consistently higher than control. The accumu-lated ammonia was generally greater in response to metrib-uzin than the other herbicides. Metribuzin inducedsignificant increases in both species throughout the wholeexperiment. However, butachlor caused significantincreases only during the first 8 days. Chlorimuron-ethylinduced a similar significant increase in wheat and maize 012340 4 8 12 16    N   i   R  a  c   t   i  v   i   t  y   (  m  g  n   i   t  r   i   t  e  c  o  n  s  u  m  e   d  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) (A)Days after treatment 02460 4 8 12 16 (B) Days after treatment Control Metribuzin Butachlor Chlorimuron-ethyl 02460 4 8 12 16    N   R  a  c   t   i  v   i   t  y   (  m  g  n   i   t  r   i   t  e  p  r  o   d  u  c  e   d  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) (A) 024680 4 8 12 16 (B) Fig. 2. Changes in activities of nitrate reductase (NR) and nitrite reductase (NiR) of (A) wheat and (B) maize shoots as a result of treatment with therecommended field dose of metribuzin, butachlor and chlorimuron-ethyl. Data are means (±SD) of at least six replications from two independentexperiments. Vertical bars represent LSD at 5% level. M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18  11  up to the 8th day and the 12th day, respectively. Soluble-Nin both species was also increased by metribuzin, butachlorand chlorimuron-ethyl during the first 12, 8 and 4 days,respectively. On the contrary, total-N content showed pro-gressive reduction following herbicide treatments. Themagnitude of reduction was most pronounced with metrib-uzin, which induced significant reductions in both speciesthroughout the whole experiment. Whilst butachlor signif-icantly decreased total-N content up to the 8th day of treat-ment whereas chlorimuron-ethyl resulted in reductions upto the 8th day and the 4th day in wheat and maize,respectively.In Table 1, concentrations of most aliphatic amino acidsin both species were increased during the first 4 days fol-lowing treatments with all herbicides. These increases weregenerally continued up to the end of the experiment inmetribuzin-treated seedlings but mostly leveled off in buta-chlor- and chlorimuron-ethyl-treated seedlings on the 8thday and the 4th day, respectively. However, proline wasdecreased mostly by chlorimuron-ethyl during the first8 days. To a lesser extent, the aromatic amino acids trypto-phan, tyrosine and phenylalanine responded in a similarpattern to aliphatic amino acids (Table 2). Metribuzin gen-erally increased the concentrations of the three aromaticamino acids throughout most time of the experiment.Slight increases were also detected following butachlortreatment during the first 4 days. On the contrary, chlo-rimuron-ethyl seemed significantly reductive to aromaticamino acids particularly during the first few days. In partic-ular, the most remarkable changes in both species were thesignificant decreases in the branched-chain amino acidsvaline, leucine and isoleucine only by chlorimuron-ethyl(Table 3). The magnitude of decrease was greater at thestart and retracted thereafter to reach control levels. Incontrast to chlorimuron-ethyl, metribuzin generallyincreased the concentrations of these amino acids duringmost of the experiment whereas butachlor showedincreases only during the first few days. Table 4 representsgreat increases in total pool size of amino acids by the threeherbicides. These increases appeared either consistent 4 8 12 16 (A)    G   S  a  c   t   i  v   i   t  y   (  m  g  g   l  u   t  a  m  y   l   h  y   d  r  o  x  a  m  a   t  e  r  e   l  e  a  s  e   d  m  g   -   1    p  r  o   t  e   i  n   h   -   1    ) 4 8 12 16(B) Control Metribuzin Butachlor Chlorimuron-ethyl 4 8 12 16 (A)    G   O   G   A   T  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 4 8 12 16 (B) Days after treatment Fig. 3. Changes in activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) of (A) wheat and (B) maize shoots as a result of treatmentwith the recommended field dose of metribuzin, butachlor and chlorimuron-ethyl. Data are means (±SD) of at least six replications from two independentexperiments. Vertical bars represent LSD at 5% level.12  M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18
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