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

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PESTICIDE Biochemistry & Physiology

Pesticide Biochemistry and Physiology 90 (2008) 8–18 www.elsevier.com/locate/ypest

Effect of metribuzin, butachlor and chlorimuron-ethyl on amino acid and 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 2007 Available online 31 July 2007

Abstract Application of the recommended field dose of metribuzin, butachlor and chlorimuron-ethyl to 10-days-old wheat and maize seedlings differentially reduced shoot fresh and dry weights during the following 16 days. Metribuzin was the most reductive while butachlor was the least. The herbicides slightly affected the activities of nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NiR, EC 1.7.7.1) but greatly inhibited glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.7.1) activities. Meanwhile, there were significant accumulations of ammonia and soluble-N accompanied by diminutions in total-N and protein contents; metribuzin exerted the 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, particularly metribuzin, cause a shortage in ammonia assimilation and subsequently a decrease in protein formation. Moreover, the elevation of soluble-N and amino acids appeared to result from breakdown of the pre-existing protein, a state that seemed consistent in seedlings treated 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 primary and secondary metabolism in crops when given to control undesired weeds. Metribuzin [4-amino-6-tert-butyl-4,5dihydro-3-methylthio-1,2,4-triazin-5-one], a triazinone herbicide, inhibits photosynthesis [1]. It interferes with photosynthetic electron transport between the primary and secondary acceptor of PSII [2]. Thus CO2 assimilation is decreased leading to starvation and reactive oxygen species are formed causing oxidative stress [3,4]. Butachlor [N-butoxymethyl-(2-chloro-2,6-diethylacetanilide], the chloroacetanilide herbicide, affects seed germination, lipid metabolism, pigment and gibberelic acid synthesis, cell division, cell permeability, mineral uptake and disturb the absorption and incorporation of amino acids into protein

*

Corresponding author. Fax: +20 57 2403868. E-mail address: [email protected] (M.M. Nemat Alla).

0048-3575/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.07.003

[5,6]. The sufonylurea herbicide, chlorimuron-ethyl [Ethy2-(((((4-chloro-6-methoxypyrimidin-2-yl)amino)carbonyl) amino) sufonyl) benzoate], inhibits acetohydroxyacid synthase (AHAS), the key enzyme for biosynthesis of valine, leucine and isoleucine [7,8]. These herbicides have varied modes of action, however, they could interfere with nitrogen cycle either directly or indirectly. Ammonia is regarded as very important for plant survival; however, its accumulation would be harmful. Among the major processes liberating ammonia is the reduction of nitrate and nitrite by NR and NiR, respectively [9]. Then ammonia is assimilated into an organic form by a two-step reaction. In the first step, glutamine is produced from the amination of L-glutamate by GS while in the second, two molecules of L-glutamate are produced from the reaction of glutamine and 2-oxoglutarate by GOGAT [10,11]. So, the changes of these activities might disturb the synthesis of amino acids and protein. Many plant species could tolerate such modifications. Therefore, the present work was aimed to ascertain the differential tolerance of wheat and maize to

M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18

metribuzin, butachlor and chlorimuron-ethyl through checking 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) and maize (Zea mays V.S.C.129) were surface sterilized by immersing in 3% sodium hypochlorite solution for 10 min, thoroughly washed, soaked for 8 h and germinated in quartz sand in plastic pots (25 cm diameter · 20 cm height). Each pot contained five germinating seeds. The pots were kept in a glasshouse at 22/10 or 28/14 C day/ night for wheat or maize, respectively, under a 14-h photoperiod at 450–500 lmol m 2 s 1 photosynthetic photon flux density, and 75–80% relative humidity. When seedlings were 10-days-old, one-fourth strength Hoagland solution was used instead of water and pots were divided into four groups. One was left to serve as control and one for each herbicide 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 quantity of each herbicide was calculated in relation to the surface area per pot and solubilized in a suitable amount of water enough to spray the surface area of each pot in one direction and crosswise. Shoots of both species were collected just before herbicide application (zero time) and also after 4, 8, 12 and 16 days from treatments, rinsed with copious amounts of water and dried by plotting with paper towels. 2.2. Assay of NADH–nitrate reductase [NADH–NR, EC 1.6.6.1]

9

33 mM KH2PO4/K2HPO4 (pH 7.5), 2 mM KNO3, 1 mM methyl viologen and 11.6 mM sodium dithionite [14]. After incubation at 30 C for 20 min in open tubes, the reaction was stopped by vigorous shaking. The diazo-coupling reagents: 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 at 540 nm to measure the nitrite lost. 2.4. Assay of glutamine synthetase [GS, EC 6.3.1.2] According to Lea et al. [10], fresh weight (10 g) was extracted in 50 mM Tris–HCl (pH 7.8) containing 1 mM sodium glutamate and 10% ethandiol (v/v). The homogenate was centrifuged at 15,000g for 15 min. Assay of GS was performed in 50 mM glutamate, 5 mM hydroxylamine hydrochloride, 50 mM MgSO4 and 20 mM ATP in 100 mM Tris–HCl (pH 7.8). After incubation at 35 C for 1 h, the reaction was terminated by ferric chloride reagent (0.67 M FeCl3, 0.37 M HC1 and 20% trichloroacetic acid, TCA, v/v). The absorbance was read at 540 nm to measure the c-glutamylhydroxamate formed. 2.5. Assay of glutamate synthase [NADH–glutamine oxo glutarate amino transferase, NADH–GOGAT, EC 1.4.7.1] Fresh weight (10 g) was extracted in 50 mM KH2PO4/ K2HPO4 (pH 7.5) containing 10 mM KCl, 5 mM EDTA, 12.5 mM b-mercaptoethanol, 1 mM PMSF, 2 mM 2-oxoglutarate, 20% ethandiol (v/v) and 0.05% Triton X-100 (w/v) [15]. The assay mixture contained 100 mM KH2PO4/K2HPO4 (pH 7.5), 0.1 mM NADH, 10 mM glutamine, 10 mM 2-oxoglutarate [16]. The reaction was followed up at 340 nm for about 10 min at 30 C to measure the consumption of NADH.

Extraction and assay were carried out according to Nakagawa et al. [12]. Plant tissue (10 g) was homogenized in 250 mM KH2PO4/K2HPO4 (pH 8.0) containing 1 mM phenyl methyl sulfonyl fluoride (PMSF), 5% isopropyl alcohol, 1 mM b-mercaptoethanol, 1 mM EDTA, 5 mM KNO3, 0.02 mM FAD and 6.3% polyclar AT (w/v). The homogenate was centrifuged at 10,000g for 15 min. The reaction mixture contained 25 mM KH2PO4/K2HPO4 (pH 7.5), 5 mM KNO3 and 0.1 mM NADH was incubated at 35 C for 30 min. The diazo-coupling reagents: 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 at 540 nm to measure the nitrite produced.

2.6. Determination of nitrogenous constituents

2.3. Assay of nitrite reductase [NiR, EC 1.7.7.1]

2.7. Determination of protein content

Fresh tissue (10 g) was homogenized in 50 mM Tris– HCl buffer (pH 7.9), containing 5 mM cysteine, 2 mM EDTA, 10 mM b-mercaptoethanol, 10% glycerol and 5% polyclar AT (w/v) [13]. The reaction mixture contained

The extraction was carried out in 80 mM Tris–HCl, pH 7.4 [20]. After centrifugation at 14,000g for 5 min, chilled 10% TCA (w/v) in acetone was added to precipitate protein over night at 4 C. Protein pellets were separated

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 solution, hypochlorite (chlorine water, saturated solution) and 8% phenol solution [18]. The contents were placed in a steam bath for 3 min then removed; cooled rapidly and 3 M NaOH was added. After 5 min, the absorbance was read at 625 nm. Both soluble-N in the water extract (3 ml) and total-N in the dried tissue (50 mg) were digested and converted into ammonia distillate using the conventional micro-Kjeldahl method [19] and determined using the indophenol method as described above.

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M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18

by centrifugation at 12,000g for 15 min, washed with chilled acetone, allowed to dry in air and reconstituted in the buffer. Protein was determined using Coomassie brilliant blue G-250 at 595 nm [21].

tion B (acetonitrile:water, 60:40), the flow rate (0.8 ml min 1), the gradient flow was performed with respect to solution B (10% at start, 6 min to 12.5%, 32 min to 58%, 33 min step 100% and 12 min wash for re-equilibration at 10% again). All values are means of at least six determinations from two independent experiments. The full data were statistically analyzed using the least significant differences (LSD) test at 5% level [24].

2.8. Determination of amino acid concentrations According to Rhodes et al. [22], amino acids were extracted with methyl alcohol and phase separated by chloroform and distilled water. The aqueous phase was dried, redissolved in water and eluted on Dowex 50-H+ with NH4OH. 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 the mixture (methanol:water:TEA:phenylisothiocyanate, 7:1:1:1) [23]. After dryness, 5 mM NaH2PO4/Na2HPO4 (pH 7.6) containing 5% acetonitrile (v/v) were added and injected into the HPLC. The system was adjusted as follows: solution A (140 mM sodium acetate containing TEA and adjusted to pH 6.4 with glacial acetic acid), soluControl

Metribuzin

3. Results Application of the recommended field dose of metribuzin, butachlor and chlorimuron-ethyl to 10-days-old wheat and maize seedlings resulted in differential significant decreases in shoot fresh weight below the control values (Fig. 1). The significant reduction in both species was continued by metribuzin up to the end of the experimental period (16 days after treatment). However, the reduction by butachlor or chlorimuron-ethyl seemed to be leveled off Butachlor 1200

Shoot fresh weight (mg plant-1)

(A)

(B)

450

800 300

400

150

0

0 0

4

8

12

16

0

120

(A) Shoot dry weight (mg plant-1)

Chlorimuron-ethyl

4

8

12

16

8

12

16

(B)

60 80 40

40

20

0

0 0

4

8

12

Days after treatment

16

0

4

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.

M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18

zin. The inhibition in GS activity was detected up to the 12th day from metribuzin treatment. Both butachlor and chlorimuron-ethyl resulted in a significant inhibition during the first 4 days in wheat and 8 days in maize. Similarly, GOGAT activity of both species was significantly inhibited by metribuzin during the whole experiment. To a lesser extent, butachlor and chlorimuron-ethyl exerted significant inhibition in the enzyme activity of both species during the first 4 days; the inhibition was extended up to the 8th day either in wheat by butachlor or in maize by chlorimuronethyl. In Fig. 4 all herbicides induced much accumulations of ammonia consistently higher than control. The accumulated ammonia was generally greater in response to metribuzin than the other herbicides. Metribuzin induced significant increases in both species throughout the whole experiment. However, butachlor caused significant increases only during the first 8 days. Chlorimuron-ethyl induced a similar significant increase in wheat and maize

after 8 days of treatment in wheat and 4 days in maize. In the same pattern, the herbicides markedly reduced dry matter; metribuzin was much more reductive than butachlor and chlorimuron-ethyl. The effect of metribuzin extended during the entire experimental period whereas butachlor and chlorimuron-ethyl showed their significant reductions only during the first 4 days from treatment. The results depicted in Fig. 2 show that NR activity was significantly increased by metribuzin up to the 12th day in wheat and the 8th day in maize relative to the untreated controls. However, butachlor and chlorimuron-ethyl caused significant decreases up to the 8th day in wheat and the 4th day in maize. On the contrary, there was a significant increase in NiR activity of both species by the three herbicides during the first 4 days of the experiment. Thereafter, the increases, if any, became non-significant. As can be seen from Fig. 3 activities of GS and GOGAT were significantly inhibited by herbicide application, the magnitude of inhibition was most pronounced by metribu-

Control

Metribuzin

NR activity (mg nitrite produced -1 -1 mg protein h )

6

Butachlor 8

(A)

Chlorimuron-ethyl

(B)

6 4 4 2 2

0

0 0

4

8

12

4

NiR activity (mg nitrite consumed -1 -1 mg protein h )

11

0

16

6

(A)

4

8

12

16

8

12

16

(B)

3

4 2

2 1

0

0 0

4

8

12

Days after treatment

16

0

4

Days after treatment

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 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.

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M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18

8 days. To a lesser extent, the aromatic amino acids tryptophan, tyrosine and phenylalanine responded in a similar pattern to aliphatic amino acids (Table 2). Metribuzin generally increased the concentrations of the three aromatic amino acids throughout most time of the experiment. Slight increases were also detected following butachlor treatment during the first 4 days. On the contrary, chlorimuron-ethyl seemed significantly reductive to aromatic amino acids particularly during the first few days. In particular, the most remarkable changes in both species were the significant decreases in the branched-chain amino acids valine, leucine and isoleucine only by chlorimuron-ethyl (Table 3). The magnitude of decrease was greater at the start and retracted thereafter to reach control levels. In contrast to chlorimuron-ethyl, metribuzin generally increased the concentrations of these amino acids during most of the experiment whereas butachlor showed increases only during the first few days. Table 4 represents great increases in total pool size of amino acids by the three herbicides. These increases appeared either consistent

up to the 8th day and the 12th day, respectively. Soluble-N in both species was also increased by metribuzin, butachlor and chlorimuron-ethyl during the first 12, 8 and 4 days, respectively. On the contrary, total-N content showed progressive reduction following herbicide treatments. The magnitude of reduction was most pronounced with metribuzin, which induced significant reductions in both species throughout the whole experiment. Whilst butachlor significantly decreased total-N content up to the 8th day of treatment whereas chlorimuron-ethyl resulted in reductions up to the 8th day and the 4th day in wheat and maize, respectively. In Table 1, concentrations of most aliphatic amino acids in both species were increased during the first 4 days following treatments with all herbicides. These increases were generally continued up to the end of the experiment in metribuzin-treated seedlings but mostly leveled off in butachlor- and chlorimuron-ethyl-treated seedlings on the 8th day and the 4th day, respectively. However, proline was decreased mostly by chlorimuron-ethyl during the first

Control

Metribuzin

GS activity (mg γ- glutamylhydroxamate released mg-1 protein h-1)

0.2

Chlorimuron-ethyl

0.3

(A)

(B)

0.15

0.2 0.1

0.1 0.05

0

0 0

4

0.12

GOGAT activity (Decrease in absorbance mg-1 protein h-1)

Butachlor

8

12

0

16

4

0.12

(A)

0.08

0.08

0.04

0.04

8

12

16

(B)

0

0 0

4

8

12

16

Days after treatment

0

4

8

12

16

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 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.

M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18

Control

Metribuzin

-1

Ammonia (mg g dry weight)

10

Butachlor

8

8

6

6

4

4

2

2

0

Soluble-N (mg ammonia g-1 dry weight)

(B)

0 0

4

8

12

30

16

(A)

0

4

8

12

60

20

40

10

20

16

(B)

0

0 0

4

8

12

80

Total-N (mg ammonia g-1 dry weight)

Chlorimuron-ethyl

10

(A)

13

16

0

4

8

12

120

(A)

16

(B)

60 80 40 40 20

0

0 0

4

8

12

Days after treatment

16

0

4

8

12

16

Days after treatment

Fig. 4. Changes in ammonia-N, soluble-N and total-N contents 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.

throughout the entire experiment with metribuzin or restricted only to the first few days with butachlor (8 days) and chlorimuron-ethyl (4 days). Fig. 5 shows that, the three herbicides resulted in significant decreases in protein content of wheat and maize

shoots. In general, metribuzin was the greatest reductive, the reduction appeared consistent and continued up to the end of the experiment. Butachlor provoked its significant reductions during the first 8 days in both species while chlorimuron-ethyl exerted the reduction up to the

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M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18

Table 1 Changes in concentration of aliphatic amino acids in wheat and maize shoots following herbicide treatments (nmolg-1 fresh tissue)a Days after treatment 0

4

8

12

Conb

Met

But

Chl

Con

Met

But

Chl

Con

16 Met

But

Chl

Con

Met

But

Chl

Wheat Asp Asn Glu Gln Ser Cys Gly His Thr Ala Pro Met Arg Lys

25.3 30.5 17.8 21.6 17.5 21.6 20.2 12.3 14.4 18.5 16.4 13.0 7.2 14.7

23.1 30.0 17.9 19.5 15.8 17.2 20.8 10.2 13.2 21.1 14.2 12.5 7.3 13.9

32.3* 39.1* 19.9 38.1* 24.5* 28.1* 27.6* 17.8* 17.1* 22.7 18.4* 16.9* 8.0 16.5*

27.4* 40.3* 15.6 35.0* 22.1* 27.0* 29.4* 12.3 19.7* 21.4 19* 15.1* 8.7 17.1*

28.1* 45.7* 16.3 35.6* 26.4* 19.2 28.2* 11.1 19.4* 21.8 11.9* 14.3* 8.9 18.8*

26.6 32.0 18.7 22.6 18.3 22.6 21.2 12.9 14.1 19.4 17.2 13.6 7.1 15.4

35.3* 39.7* 24.0* 34.7* 25.6* 29.7* 27.5* 19.2* 18.8* 24.9* 23.3* 16.1* 8.6 20.2*

30.0* 38.1* 21.1 29.5* 20.6 25.5* 23.9 15.6* 17.0* 21.8 19.5* 15.1 8.5 21.1*

28.6 35.4* 20.1 26.3* 19.7 24.3 21.8 11.9 13.2 17.9 14.5* 14.7 8.1 20.6*

25.3 30.5 17.8 21.6 18.5 21.6 20.2 12.3 14.4 18.5 16.4 13.0 7.2 14.7

33.7* 40.5* 23.7* 28.7* 20.2 28.7* 26.9* 13.1 19.1* 24.6* 18.2 17.3* 9.6* 15.6

28.2* 36.9* 19.8 26.1* 19.4 26.4* 22.5 13.7 16.0 20.6 18.3 14.5 8.0 16.1

26.0 31.3 18.3 22.1 17.9 22.1 20.7 12.6 14.8 19.0 16.9 13.3 7.4 15.1

26.7 32.2 18.8 22.8 18.4 22.8 21.3 13.0 15.2 19.5 17.3 13.7 7.6 15.5

27.3 37.3* 19.7 26.4* 17.4 26.4* 24.8* 12.1 17.6* 22.7* 16.1 15.9* 8.8* 16.3

27.6 33.2 19.4 23.5 19.0 23.5 22.0 13.4 15.6 20.1 17.9 14.2 7.8 16.0

28.3 34.6 16.5 20.4 19.1 21.4 22.3 12.1 14.2 22.1 16.1 13.5 7.5 14.3

Maize Asp Asn Glu Gln Ser Cys Gly His Thr Ala Pro Met Arg Lys

39.7 45.9 34.7 38.6 39.7 24.1 27.4 25.7 22.9 28.5 24.1 17.9 17.4 23.5

41.5 45.9 36.2 40.3 41.5 25.1 28.6 26.9 23.9 29.8 25.1 18.7 18.1 24.5

47.1* 68.4* 40.1 57.8* 57.1* 26.3 35.5* 27.8 29.2* 33.7 29.5* 21.2* 23.6* 27.9*

45.1 62.1* 39.4 48.2* 49.1* 27.3 31.2 28.2 26.1 36.4* 27.3* 20.3 19.7 26.7

49.9* 65.3* 39.9 52.9* 51.9* 33.0* 33.1* 29.1 25.4 34.4* 21.1* 26.6* 19.9 29.3*

42.0 48.5 36.7 40.8 42.0 25.4 29.0 27.2 24.2 30.2 25.4 18.9 18.3 24.8

50.7* 64.6* 44.3* 59.2* 50.7* 30.7* 39.5* 37.9* 25.3 30.4 33.7* 22.9* 20.3 30.6*

48.6* 59.1* 42.5* 47.8* 48.6* 29.4* 33.6* 31.5* 26.1 34.9* 27.4 21.9 21.2* 28.8*

43.5 58.2* 39.8 49.2* 43.5 26.3 33.2 29.2 28.1* 33.2 27.3 20.6 19.0 6.7

40.8 47.2 37.7 39.7 40.8 24.7 28.2 27.5 23.6 29.3 24.7 18.4 17.8 24.2

43.6 57.9* 40.2 48.7* 50.2* 30.4* 35.4* 30.5 22.6 36* 25.1 22.6* 16.9 26.4

42.0 48.5 41.8 48.8* 42.0 25.4 33.5* 27.2 24.2 30.2 25.4 18.9 21.3* 24.8

41.5 47.9 36.2 40.3 37.5 25.1 28.6 24.9 24.0 26.8 23.1 18.7 18.1 26.6

41.1 47.5 35.9 40.0 41.1 24.9 28.4 29.7 23.8 29.5 24.9 18.5 18.0 24.3

42.1 56.7* 42.9* 41.9 42.5 23.9 30.2 31.8 24.6 35.3* 22.4 22.1 21.4* 29*

41.5 48.0 36.3 37.4 38.5 25.1 29.7 28.9 24.0 31.8 25.1 16.7 17.1 22.6

45.3 49.7 36.0 43.1 43.3 25.9 28.5 28.7 24.8 26.6 22.5 17.6 16.8 22.4

a b

Values of treated samples followed by an asterisk are significantly different at 5% level with respect to untreated control. Con, control; Met, metribuzin; But, butachlor; Chl, chlorimuron-ethyl.

8th day and the 4th day in wheat and in maize, respectively. 4. Discussion In general, herbicides kill plants by disrupting essential physiological or biochemical process, usually through a specific interaction with a single molecular target in the plant [25]. The present results generally showed a great reduction in fresh and dry weights of wheat and maize shoots as a result of treatment with metribuzin, butachlor and chlorimuron-ethyl although varied in their modes of action. However, both species seemed to tolerate butachlor more than metribuzin or chlorimuronethyl, to some extent. Growth reduction of several plant species was reported after the application of triazine and triazinone herbicides [26,27], a-chloroacetanilides [28–31] and sulfonylureas [31,32]. Thus, the differential influence of herbicides could result from varied disturbances in certain processes, e.g., those related to nitrogen metabolism. Therefore, the nitrogenous constituents, concentrations of free amino acids and protein content as well

as activities of some nitrogen-related enzymes were checked. For its role in nitrogen and protein biosynthesis, ammonia is considered as very important for plant survival if it could be safely utilized by the plant cell. However, its accumulation would be harmful to plant tissues. The present results revealed an elevation of ammonia and soluble-N following herbicide treatments concomitantly with an inhibition in growth, total-N and protein contents. Consequently, growth reduction might result from malfunction of nitrogen metabolism. In accordance, Kaiser et al. [33] found that the primary products of nitrate and nitrite reduction are potentially toxic. Scarponi et al. [34] found an increase in ammonia in broad bean treated with propachlor, chlorimuron-ethyl and imazethapyr. The relationship between ammonia elevation and growth reduction was evident. Thus, the reduction in growth might result from the over accumulation of ammonia that might exceed the safe levels to be toxic. On the other hand, the increase in ammonia accompanied by the decrease in both total-N and protein could point to a shortage in ammonia assimilation and conse-

M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18

15

Table 2 Changes in concentration of aromatic amino acids in wheat and maize shoots following herbicide treatments (nmol g-1 fresh tissue)a Days after treatment

Wheat b

Maize

Con

Met

But

Chl

Con

Met

But

Chl

0 4 8 12 16

17.5 19.2 18.3 17.5 18.4

21.5 23.6* 23.2* 21.4*

20.3 20.4 19.4 19.0

17.3* 19.7 17.9 16.1

34.1 35.6 36.1 35.1 35.3

43.5* 43.6* 33.1 32.2

44.2* 41.8* 36.1 33.7

31.2* 37.3 32.7 32.5

0 4 8 12 16

11.0 13.9 11.5 11.0 11.6

14.3 13.5* 12.3 13.4*

14.7 13.2* 12.2 11.9

11.5* 10.9 11.2 10.4

23.5 24.5 24.8 24.2 24.3

25.9 27.1 29.7* 29.0*

33.7* 28.8* 24.8 26.6

23.3 28.7* 21.6 21.4

0 4 8 12 16

9.2 12.2 9.7 9.2 9.8

14.3 13.4* 12.3* 7.3

14.7 12.9* 10.3 10.1

10.5* 10.4 9.5 9.6

11.8 12.3 12.4 12.1 12.2

16.9* 17.0* 11.5 14.5*

13.4 14.4* 16.4* 12.3

11.2 12.9 10.3 10.9

Tyr

Phe

Try

a b

Values of treated samples followed by an asterisk are significantly different at 5% level with respect to untreated control. Con, control; Met, metribuzin; But, butachlor; Chl, chlorimuron-ethyl.

Table 3 Changes in concentration of branched-chain amino acids in wheat and maize following herbicide treatments (nmol g Days after treatment

Wheat b

1

fresh tissue)a

Maize

Con

Met

But

Chl

Con

Met

But

Chl

0 4 8 12 16

14.4 16.5 15.1 14.4 15.2

17.1 17.8* 19.1* 17.6*

23.3* 17.1 16.0 15.6

8.4* 10.2* 13.0 14.6

17.9 18.7 18.9 18.4 18.5

20.2 24.9* 22.6* 22.1*

23.3* 19.4 18.9 20.1

9.5* 13.6* 15.7 16.6

0 4 8 12 16

15.8 17.5 16.5 15.8 16.6

19.6 19.5* 17.2 17.3

24.1* 15.7 17.5 17.1

7.8* 12.8* 14.4 14.3

22.9 23.9 22.2 23.6 23.8

29.2* 22.5 25.8 28.3*

28.1* 19.1 24.2 24.0

13.8* 17.1* 22.2 20.2

0 4 8 12 16

13.0 11.6 13.6 13.0 13.9

17.4 17.6* 16.1* 15.1* 15.2

18.5* 15.6 14.5 14.2

5.2* 10.7* 11.2 12.5

18.1 18.3 17.8 18.0

21.1 22.1* 20.1 20.1

19.7 19.9 18.3 18.1

12.1* 15.3* 15.9 17.4

Val

Ile

Leu

a b

Values of treated samples followed by an asterisk are significantly different at 5% level with respect to untreated control. Con, control; Met, metribuzin; But, butachlor; Chl, chlorimuron-ethyl.

quently a decrease in synthesis of both amino acids and protein. In addition, the elevation of amino acids could explain the rise in soluble-N content. Moreover, the increase in soluble-N concomitant with either decreases in total-N and protein contents or increases in most amino acids could suggest an increase in proteolysis. These findings revealed that the increase in amino acid concentrations was not a result of an increased synthesis rate but might be

produced from an enhanced lysis of protein. Evidences for increase in proteolysis in plants have been indicated in response to biotic and abiotic stresses [35,36]. Stressed plants have altered nitrogen metabolism and increased protease activity [36,37]. It was observed that stress increased protease activity and amino acid content in non-tolerant lines but not in tolerant lines [38]. Therefore, a drop of protein content is predictable to be a resultant of a decrease in

16

M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18

Table 4 Changes in concentration of total pool size of free amino acids in wheat and maize following herbicide treatments (nmol g Days after treatment

Wheat Con

a b

fresh tissue)a

Maize

b

0 4 8 12 16

1

Met

331.9 327.6 346.4 332.9 350.3

But

431.4* 451.5* 419.1* 381.5*

425.6* 402.2* 376.4 361.1

Chl

Con

Met

But

Chl

366.4* 351.8 334.7 339.9

537.7 559.2 566.1 555.8 559.7

681.0* 698.0* 629.3* 613.0*

649.5* 644.8* 592.7 557.5

612.9* 582.7 537.7 550.2

Values of treated samples followed by an asterisk are significantly different at 5% level with respect to untreated control. Con, control; Met, metribuzin; But, butachlor; Chl, chlorimuron-ethyl.

Control

Metribuzin

Butachlor

Chlorimuron-ethyl

Protein content (mg g-1 fresh weight)

(A)

(B)

8

8

6

6

4

4

2

2

0

0 0

4

8

12

Days after treatment

16

0

4

8

12

16

Days after treatment

Fig. 5. Changes in protein contents 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.

de novo synthesis and an increase in breakdown of the preexisting molecules. So, the changes in protein turnover rate might give rise to increases of most amino acids and soluble-N. However, this was not the case for valine, leucine and isoleucine. As the other sulfonylurea herbicides, chlorimuron-ethyl inhibits AHAS, the key enzyme for biosynthesis of these branched-chain amino acids. These results are in conformity with other investigations [22,39,40]. Thus, Rhodes et al. [22] hypothesized that the increases in free amino acid levels in Zea mays and in Lemna minor treated with AHAS inhibitors were due to degradation of pre-existing proteins rather than to new amino acid synthesis. Similarly, Nemat Alla and Hassan [39] concluded that rimsulfuron increased the rate of protein resolution than amino acid synthesis in soybean. Among the different routes liberating ammonia are the reductions of nitrate and nitrite through the action of NR and NiR. The present results indicate that NR was increased by metribuzin but decreased by butachlor or chlorimuron-ethyl. On the other hand, NiR activity was increased by the three herbicides during the first few days. These findings claimed that the NR–NiR system was not greatly modified. In spite of enhancing NR and NiR activ-

ities, metribuzin in particular decreased protein content concluding that ammonia starvation did not seem as the cause of protein synthesis inhibition but probably a shortage in its assimilation into organic forms. Ammonia assimilation takes place in plants by a two-step combined reaction catalyzed by GS and GOGAT [10,11,34] forming glutamate which can serve as a nitrogen source for biosynthesis of many other amino acids. The present results indicate a remarkable inhibition in GS and GOGAT activities by herbicides accompanied with a decrease in protein content. Thus, the failure in GS–GOGAT system function would cause over accumulation of unassimilated ammonia and consequently could lead to diminution in amino acid formation with a subsequent drop in protein synthesis. Devine and Preston [25] indicated that inhibition of GS would lead to inhibition in amino acid synthesis. This could confirm that the elevation of amino acids was not due to an increased rate of synthesis. Actually, there was a relationship between the increase in ammonia and the inhibition in GS and GOGAT activities. Meanwhile there were rises in most amino acids accompanied with decreases in protein content. These findings conclude that these increases were not resulted from an efficient organization

M.M. Nemat Alla et al. / Pesticide Biochemistry and Physiology 90 (2008) 8–18

of ammonia but might result from protein lysis. The effect of metribuzin seemed the worst. It binds to the D1 protein in PSII. Upon binding, the electron flow from PSII is disrupted, and carbon dioxide fixation ceases and free radicals are generated and chlorosis develops [41,42]. Moreover, a shortage in incorporation of amino acids could reduce protein synthesis. Thus, not only the increase in protein degradation but also the retardation of amino acid incorporation could drop protein content. In fact, the incorporation of amino acids in the process of protein synthesis has been affected by several herbicides. Egli et al. [43] reported that the inhibition of protein synthesis by herbicides is primarily due to disturbance of the absorption and retention of amino acids available for protein synthesis, interference with the incorporation of amino acids into protein, and/or the formation of enzymes responsible for protein synthesis and metabolism. Duke et al. [44] detected reductions in protein synthesis in the hypocotyls of soybean and cucumber by a-chloroacetanilide herbicides several hours before the observed inhibition of growth concluding decreases in the incorporation of leucine. Also, Moreland et al. [5] indicated that propachlor inhibited leucine incorporation into protein in soybean hypocotyls. Moreover, Jaworski [45] suggested that because of its reactive nature, the a-halogen of the a-chloroacetanilide herbicides undergoes a nucleophilic displacement with the amino group of methionyl-tRNA. This would prevent peptide chain linkage from occurring since methionyl-tRNA may act as peptide chain inhibitor in certain plants. Therefore, the decrease in protein content by the three herbicides, in the present work, was not surprising in spite of the varied modes of action. The effects of butachlor and metribuzin on nitrogen metabolism are secondary; however, both herbicides undergo nucleophilic displacement attacking thus protein synthesis. On the other hand, chlorimuronethyl had a direct relationship with nitrogen metabolism. It inhibits the activity of AHAS enzyme reducing therefore the branched-chain amino acids and consequently leading to drop in protein synthesis. 5. Concluding remarks The present results indicate a state of stress induced in wheat and maize seedlings by the recommended field dose of metribuzin, butachlor or chlorimuron-ethyl with differential phytotoxicity. Growth, nitrogen fractions, amino acid concentrations, protein content and activities of NR, NiR, GS and GOGAT were variably influenced. Metribuzin seemed the most reductive to growth whereas butachlor was the least. The changes in all parameters by butachlor and, to some extent, chlorimuron-ethyl were restricted to the first few days then recovered thereafter while the effect of metribuzin continued. These results revealed that both species tolerated the effects of butachlor rather than metribuzin with intermediary tolerance to chlorimuron-ethyl. The differential tolerance to herbicides appeared to relate with recoveries in changes of nitrogenous fractions and enzyme

17

activities. The three herbicides, particularly metribuzin, led to accumulation in ammonia and soluble-N whereas total-N and protein were depressed. However, there were little changes in NR and NiR activities concomitant with many inhibitions in GS and GOGAT activities indicating that the drop in protein might not be attributable to ammonia demand but probably to decreases in its assimilation. Nevertheless, most of the individual amino acids were elevated; however, there was a decrease in valine, leucine and isoleucine of both species only by chlorimuron-ethyl. The increases in amino acids seemed to result from a change in turnover rate of the pre-existing protein rather than from new syntheses. These findings reveal to a drop in protein synthesis and rise in its lysis resulting in increased levels of soluble-N and most of amino acids. References [1] L.W. Mengistu, G.W. Mueller-Warrant, A. Liston, R.E. Barker, psb mutation (valine219 to isoleucine) in Poa annua resistant to metribuzin and diuron, Pest Manag. Sci. 56 (2000) 209–217. [2] J.W. Gronwald, Resistance to PSII inhibiting herbicides, in: S.B. Powles, J.A.M. Holtum (Eds.), Herbicide Resistance in Plants, Lewis publishers, Boca Raton, London, Tokyo, 1994, pp. 27–59. [3] E. Kuzniak, Transgenic plants: an insight into oxidative stress tolerance mechanisms, Acta Physiol. Plant 24 (2002) 97–113. [4] M.M. Nemat Alla, N.M. Hassan, Changes of antioxidants levels in two maize lines following atrazine treatments, Plant Physiol. Biochem. 44 (2006) 202–210. [5] D.E. Moreland, S.S. Malhotra, R.D. Gruenhagen, E.H. Shokrah, Effects of herbicides on RNA and protein synthesis, Weed Sci. 17 (1969) 556. [6] P.C. Kearney, D.D. Kaufman, Herbicides: Chemistry Degradation and Mode of Action, vol. 3, Dekker, New York, 1988. [7] E. Dewaele, G. Forlani, D. Degrande, E. Nielsem, S. Rambour, Biochemical characterization of chlorsulfuron resistance in Cichorium intybus L. var Witloof, J. Plant Physiol. 151 (1997) 109–114. [8] J. Schroeder, Cucumber (Cucumis sativus) response to selected foliarand soil-applied sulfonylurea herbicides, Weed Tech. 12 (1998) 595– 601. [9] B.J. Miflin, P.J. Lea, The Biochemistry of Pants, vol. 16, Academic Press, New York, 1990. [10] P.J. Lea, S.A. Robinson, G.R. Stewart, The enzymology and metabolism of glutamine glutamate and asparagines, in: P.J. Miflin, B.J. Lea (Eds.), The Biochemistry of Plants, vol. 16, Academic Press, NewYork, 1990, pp. 121–159. [11] T.W. Goodwin, E.I. Mercer, Introduction to Plant Biochemistry, third ed., Pergamon Press, 1983. [12] H. Nakagawa, Y. Yonimura, H. Yamamota, T. Sato, N. Ogura, R. Sato, Spinach nitrate reductase: purification, molecular weight, and subunit composition, Plant Physiol. 77 (1985) 124–128. [13] S. Nagaoka, M. Hirasawa, K. Fukushima, G. Tamura, Methyl viologen-linked nitrite reductase from bean roots, Agric. Biol. Chem. 48 (1984) 1179–1188. [14] J.L. Wray, P. Filner, Structural and functional relationships of enzyme activities induced by nitrate in barley, Biochem. J. 119 (1970) 715–725. [15] A.J. Marquez, C. Avile, B.G. Forde, R.M. Wallsgrove, Ferredoxinglutamate synthase from barley leaves: rapid purification and partial characterization, Plant Physiol. Biochem. 26 (1988) 645–651. [16] U. Hecht, R. Oelmu¨ller, S. Schmidt, H. Mohr, Action of light, nitrate and ammonium on the levels of NADH- and ferridoxin-dependent glutamate synthases in the cotyledons of mustard seedlings, Planta 175 (1988) 130–138.

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