[show abstract][hide abstract] ABSTRACT: To evidence a possible mechanism of defense toward oxidative stress induced by herbicides in plants, an investigation was carried on the activity of glutathione peroxidase (GPX) in Italian ryegrass (Lolium multiflorum) and in Festuca (Festuca arundinacea) in response to atrazine (6-chloro-N-ethyl-N'-isopropyl-1,3,5-triazine-2,4-diamine) and fluorodifen (4-nitrophenyl α,α,α-trifluoro-2-nitro-p-tolyl ether). In general, the herbicide treatments significantly induced GPX activity in the shoots of Italian ryegrass, whereas inhibited it in Festuca. These opposite behaviors are examined, taking into account the accumulation and persistence of the two herbicides in the plants, and they are discussed in terms of GPX counteraction to oxidative stress in the first case, and of a lower detoxification rate unable to prevent a deleterious effect on the GPX activity in the second case. Further information on the properties of Italian ryegrass and Festuca GPX were achieved by purification and isolation of the enzymes, performed by protein liquid chromatography and by electrophoretic analyses. GPX of both the plants were found to be heterodimer with multiple function in showing also glutathione S-transferase (GST) activity.
Journal of Environmental Science and Health Part B Pesticides Food Contaminants and Agricultural Wastes 01/2011; 46(8):715-22. · 1.10 Impact Factor
[show abstract][hide abstract] ABSTRACT: The expression of glutathione S-transferase (GST) activity in Festuca arundinacea was investigated in response to the following herbicide safeners: benoxacor, cloquintocet-mexyl, fenchlorazol-ethyl, fenclorim, fluxofenim and oxabetrinil. All the above compounds enhanced the GST activity tested towards the "model" substrate 1-chloro-2,4-dinitrobenzene (CDNB). Assays of GST activity towards the herbicides terbuthylazine (N(2)-tert-butyl-6-chloro-N(4)-ethyl-1,3,5-triazine-2,4-diamine) and butachlor (N-butoxymethyl-2-chloro-2',6'-diethylacetanilide) as substrates also showed the ability of the safeners to enhance the enzyme activity towards both these herbicides, with the exception of cloquintocet-mexyl for the enzyme activity towards butachlor. As a consequence of the above effects at a macro-scale level, decreased herbicide accumulation and persistence were ascertained in response to the addition of the safener benoxacor to both terbuthylazine and butachlor treatments. These results are discussed in terms of capacity of benoxacor to induce herbicide detoxification in Festuca arundinacea with a view to utilizing them in reducing herbicide pollution.
Journal of Environmental Science and Health Part B Pesticides Food Contaminants and Agricultural Wastes 11/2009; 44(8):805-9. · 1.10 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effect of treatments with four herbicides and a safener on the activity of triosephosphate isomerase (TPI) extracted from shoots of Italian ryegrass was investigated. It was found that atrazine and fluorodifen, herbicides which interfere with photosynthesis, caused a decrease in measured enzyme activity. In addition, the in vitro effect of oxidized glutathione (GSSG), a compound produced in situations of oxidative stress, on TPI activity was investigated. It was shown that GSSG was a strong inhibitor of enzyme activity, at low concentrations in a dose-time-dependent manner. The enzyme extracts were submitted to chromatographic purifications and to two-dimensional electrophoresis. Some spots had molecular masses ranging between 20 and 30 kDa and were characterized and identified by LC-ESI-MS/MS as TPIs. The mass spectrometry also made it possible to identify the presence of cysteine residues that could be subjected to S-glutathionylation, which regulate the enzyme activity.
Journal of Agricultural and Food Chemistry 09/2009; 57(17):7924-30. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Buffer strips are uncultivated zones left along the boundaries of crops. Buffer strips are used to eliminate or reduce the
environmental impact of herbicides. As the efficiency of buffer strips is improved by the presence of non-crop vegetation,
we studied the possible role of growing the perennial grass Festuca arundinacea. We investigated the activity in festuca of glutathione S-transferase (GST), which is an enzyme very active in metabolising
herbicides. Our results evidence GST activity, which is enhanced by the following compounds: benoxacor, cloquintocet-mexyl,
fenchlorazol-ethyl, fenclorim, fluxofenim and oxabetrinil. These synthetic compounds are named herbicide safeners because
they protect crop plants against injury from some herbicides without reducing the action of herbicides against the target
weeds. The increases in GST activity were found to be concomitant with changes in Vmax and KM, that are kinetic constants related directly to the enzyme concentration in the protein “pull” and inversely to the substrate-enzyme
affinity, respectively. In particular, Vmax increase with KM decrease was observed in response to benoxacor, Vmax increases were found in response to fenchlorazol-ethyl, fenclorim, fluxofenim and oxabetrinil, and KM decrease was observed in response to cloquintocet-mexyl. The GST activity was also found to be enhanced by the safeners when
it was tested toward the herbicides terbuthylazine and butachlor as substrates. In particular, the increases in GST toward
terbuthylazine ranged in the following decreasing order: 154.6%, 91.7%, 89.2%, 88.3%, 82.5% and 30.8% in response to fluxofenim,
fenchlorazol-ethyl, fenclorim, oxabetrinil, benoxacor and cloquintocet-mexyl, respectively. The increases in GST toward butachlor
ranged in the following decreasing order: 77.0%, 71.2% 59.0%, 41.0% and 33.1% in response to oxabetrinil, benoxacor, fenclorim,
fluxofenim and fenchlorazol-ethyl, respectively. A further test, performed to evaluate the relevance of the above effects
on a macro-scale level, evidenced 10.1% and 32.7% increased amounts of metabolised terbuthylazine and butachlor, respectively,
in response to the addition of benoxacor safener to the herbicide treatments. These results indicate that herbicide diffusion
following the runoff of surface waters can be prevented or significantly reduced by vegetating buffer strips with festuca
and by the combination of herbicide and a suitable safener.
–glutathione S-transferase–terbuthylazine–butachlor–safeners–detoxification–buffer strips
Agronomy for Sustainable Development 01/2009; 29(2):313-319. · 3.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Over recent years it has emerged how certain no crop-species can be employed in phytoremediating contaminated soils or preventing herbicide pollution; in this contest Festuca arundinacea was investigated. Shoots of Festuca were submitted to fast protein liquid chromatography in order to identify their glutathione S-transferases (GST; EC 184.108.40.206), by a combination of anionic, affinity and RP-HPLC chromatography. The chromatographic procedure revealed satisfactory yield and four GSTs were identified: they were named FaGST I, FaGST II, FaGST III and FaGST IV. Among these, significant differences were observed in the chromatographic behaviours, structure, activity toward a "model" substrate, 1-chloro-2,4-dinitrobenzene, and responsiveness to the herbicide safener benoxacor. FaGST I showed the highest activity toward the above substrate, and this activity was up-regulated by the herbicide safener. Therefore, FaGST I was purified till homogeneity and was determined to be an heterodimer consisting of two subunits of 28.0 and 27.2kDa. Each subunit of FaGST I was further characterized by means of LC-ESI-MS/MS and immunoblotting analysis, which revealed that both the subunits belong to the tau subclass.
[show abstract][hide abstract] ABSTRACT: The expression of glutathione S-transferase (GST) activity in wheat and maize shoots was investigated in response to treatments with the herbicide safeners benoxacor, cloquintocet-mexyl, fenchlorazole-ethyl, fenclorim, fluxofenim and oxabetrinil. These safeners significantly enhanced the GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) as a 'standard' substrate, with the exception of oxabetrinil in maize. The enhancements of GST (CDNB) activity were found to be concomitant with increases in V(max) (the reaction rate when the enzyme is fully saturated by the substrate) in wheat following cloquintocet-mexyl and fenchlorazole-ethyl treatments, and in maize following fenchlorazole-ethyl treatment. Otherwise, decreases in V(max) were observed in wheat and maize following fenclorim and fluxofenim treatments. With the exception of oxabetrinil, all the safeners significantly reduced the apparent K(M) (the substrate concentration required for 50% of maximum GST activity) of both wheat and maize GST. The V(max) and K(M) variations following safener treatments are discussed in terms of an increased expression of GST enzymes and an increased affinity for the CDNB substrate. The activity of wheat and maize GST was also assayed towards butachlor and terbuthylazine respectively; the results indicate the ability of cloquintocet-mexyl, fenchlorazole-ethyl and fluxofenim to enhance the enzyme activity in wheat and of benoxacor and fenchlorazole-ethyl to do so in maize.
Pest Management Science 11/2006; 62(10):927-32. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Non-enzymic and peroxidase-catalysed oxidative decarboxylations of tryptophan (TPP) were studied. The in-vitro rate of enzymic reaction was affected by various herbicides at low concentrations (10−5−10−4M): dinoseb, 2,4-D, dichlobenil and benazolin acted as inhibitors; on the other hand, chlorpropham, bromacil, diphenamid and 4-ethylamino-6-isopropylamino-1,3,5-triazin-2-ol (hydroxyatrazine) were stimulatory. The results of in-vivo experiments showed that chlorpropham and 2,4-D changed the activity of peroxidase from Brassica germinated seeds in vivo, as they do in vttro. Determinations of consumed TPP were carried out either by spectro-photometry or by chromatography.
[show abstract][hide abstract] ABSTRACT: Analytical methods are described for the determination of residues of benfluralin in soil and carrots. All plots treated with 1.3 or 1.9 kg benfluralin/ha produced average carrot yields significantly higher than untreated plots. Degradation of benfluralin in soil was very slow during the winter. As the temperature rose, the rate of benfluralin decline was accelerated; 324 days after incorporation, benfluralin residues were reduced to about 11 and 14% of the applied dose. The average total residues of benfluralin found in whole carrots were 210 ± 18 and 240 ± 47 ng/g following doses of 1.3 and 1.9 kg a.i./ha respectively. The peel of carrots contained 92% of the total residue, and there seemed to be little translocation of the compound through the plant tissues.
[show abstract][hide abstract] ABSTRACT: A gas-liquid chromatographic method using electron-capture detection is described for the determination of perfluidone residues in rice grains and husks. The procedure consists of extraction, purification on an anion-exchange resin, methylation and further clean-up (if necessary) by adsorption on “Florisil”. The recoveries from fortified rice grains and husks were respectively 73.5–98% and 77–104% in the range 0.01–0.50 mg/kg. Sensitivity permits detection of perfluidone at 0.005 mg/kg level.
[show abstract][hide abstract] ABSTRACT: A procedure was developed to obtain non-embryogenic callus and regenerated lines from root segments of Zea mays grown in aseptic conditions. The activity of glutathione-S-transferases (GSTs), for non-embryogenic callus, was determined toward 1-chloro-2,4-dinitrobenzene (CDNB) and it was compared with that obtained for corn seedlings grown without hormones. For the callus masses, increases of specific activity toward CDNB and the kinetic parameter Vmax were observed with respect to corn seedlings. The procedure permitted the regenerating of tissues from callus explants, therefore the GST(CDNB) activity and the effect of the safener benoxacor on its expression were investigated for the regenerated tissues grown in agarized substrate and in liquid medium. These explants showed a constitutive GST(CDNB) activity higher than corn seedlings and this activity was increased, for both tissues, in response to the presence of the safener benoxacor in the growing medium. The GST activity for the above tissues was also assayed toward benoxacor and terbuthylazine, metolachlor and fluorodifen herbicides. Measurable GST activity was found toward some of the above chemicals and it was found to be significantly enhanced in response to benoxacor treatment.
[show abstract][hide abstract] ABSTRACT: Herbicide runoff and the effects of a narrow vegetative filter strip (VFS) were studied on an arable field in the low-lying plains of the Veneto Region (north-east Italy). Cultivated plots were compared with and without a 6m wide VFS composed of trees, shrubs and grass. Natural and simulated runoff were monitored during 2000 and 2001. Herbicides applied on the field were: metolachlor (2184-2254 g ha(-1)), terbuthylazine (1000-1127 g ha(-1)) and isoproturon (1000 g ha(-1)). The VFS reduced both runoff depth (10.2-91.2%) and herbicide losses (85.7-97.9%) in the monitored rainfall events. Total herbicide loss with runoff was low (0.69-3.98 g ha(-1) without VFS, less than 0.27 g ha(-1) with VFS), but concentrations were sometimes very high, especially of terbuthylazine and isoproturon during the first events after treatment. In these events there was a high probability of exceeding the ecotoxicological endpoint for algae, but the VFS helped to reduce the potential risk. Two VFS effectiveness mechanisms were identified: (i) dilution, and (ii) a "sponge-like" effect, which temporarily trapped chemicals inside the VFS before releasing them.
[show abstract][hide abstract] ABSTRACT: Butachlor is a chloroacetanilide herbicide successfully employed in weeding some important crops, and benoxacor is a safening compound able to induce the enzymatic mechanism of chloroacetanilide detoxification in plants. A practical method for a simultaneous detection of butachlor and benoxacor residues in wheat and in soil is described. The procedure can be performed by GC and HPLC. They were extracted with methanol and cleaned up by solid phase extraction (SPE). The analytes were satisfactorily separated via both GC and HPLC techniques, and no interferences were observed coming from plant or soil matrixes or reagents. The limit of quantitation was found to be 5.0 ng by GC and 20.0 ng by HPLC for butachlor and 2.5 ng by GC and 15.0 ng by HPLC for benoxacor. Butachlor recovery tests ranged from 85.4% to 91.7% in wheat shoots and 84.0% to 93.2% in soil; benoxacor recovery tests ranged from 86.5% to 90.8% in wheat shoots and 85.7% to 90.7% in soil. The reproducibility and the accuracy make this method a selective and sensitive tool for routine analyses.
Journal of Agricultural and Food Chemistry 07/2005; 53(11):4326-30. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The action of safener benoxacor on the detoxification of terbuthylazine (TBA) in Zea mays and Festuca arundinacea was ascertained by the investigation of the effects of benoxacor on the activity of glutathione-S-transferases (GSTs) in the shoots of the two plant species. TBA treatment generally reduced GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) in corn and did not affect the enzyme activity in festuca. When applied alone, benoxacor stimulated GST activity in both plants; however, when it was applied in mixture with TBA, generally, an enhancement of the enzyme activity was found in corn but not in festuca in comparison with the respective TBA-treated samples. The enhancement of GST activity in response to the benoxacor treatment in both corn and festuca resulted to be concomitant with decreases in apparent K(M) in both the plant species, with V(max) unaffected, and with an increased expression of proteins having molecular masses in the characteristic range of plant GSTs. After the benoxacor treatment, increased GST activity toward TBA as a substrate was observed in both corn and festuca. As a consequence, lesser amounts and persistence of TBA residues were found in shoots of corn and festuca treated with the TBA and benoxacor mixture compared to TBA-only-treated samples. Therefore, benoxacor enhances TBA detoxification in both corn and festuca; the induction of detoxifying activity in a nondomesticated grass is discussed in view of its use in vegetating buffer strips around crops to prevent TBA pollution.
Journal of Agricultural and Food Chemistry 05/2005; 53(7):2483-8. · 2.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The persistence of pretilachlor applied to rice singly or in combination with the safener fenclorim was investigated in connection with starch, glucose and protein formation. The addition of fenclorim to pretilachlor did not reduce the accumulation of the latter, but reduced its persistence in rice shoots, while the presence of pretilachlor did not affect the persistence of fenclorim, but significantly increased its accumulation in the shoots. Therefore the safening effect of fenclorim consisted of a more rapid detoxification of pretilachlor. Over the period of pretilachlor and fenclorim detoxification, decreases in starch content, partially counterbalanced by increases of free glucose, and decreases in total protein content were observed in pretilachlor-treated shoots; decreases in both starch and free glucose, as well as in total protein content, were observed in fenclorim -treated shoots compared with the untreated controls. The decreases in starch and total glucose appeared to be a direct consequence of reduced glucokinase and ribulose-1,5-bisphosphate carboxylase activity, and the decrease in total protein an indirect consequence of reduced glutamine synthetase and glutamate synthase activity, in response to pretilachlor and fenclorim treatments.
Pest Management Science 05/2005; 61(4):371-6. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: The contents and persistence of pretilachlor (P) and of the safener fenclorim (F) in rice treated with P, F and their mixture were investigated in relation to the expression of glutathione-S-transferase (GST) activity in response to treatments. F failed to reduce the accumulation of P, however it was able to reduce its persistence in rice shoots. On the other hand, while P did not reduced F persistence, it determined a greater than two-fold F accumulation in plants. Therefore, a detoxification mechanism common to the two compounds but favourable to P was hypothesised. This mechanism consists in an activation of GST activity toward P as substrate [GST(P)] in response to all treatments. The activation levels were found to be proportional to the amount of persistent P and F, and to be additive following the P-F mixture treatment. The kinetic parameters V max and K m of GST(P) were increased and decreased, respectively, in rice shoots treated with P, F and particularly with P-F mixture. Therefore, a defence mechanism toward P, consisting in an increased expression of GST(P) in rice plant, both as the global amount of the enzyme in the protein "pull" and the induction of the enzyme isoforms more sensitive to P, in response to all treatments, especially the P-F mixture treatment, is confirmed. This finding is consistent with the observed effect of F on P persistence and detoxification in the rice shoots. Oryza sativa / pretilachlor / fenclorim / persistence / glutathione-S-transferase Résumé – Persistance et détoxication du pretilachlore et du fenclorim dans le riz (Oryza sativa). On a étudié la teneur et la persistence du pretilachlore (P) et du fenclorim (F), un antidote, dans le riz traité avec P, F et un mélange des deux par référence à l'activité de la glutathion-S-transférase (GST) en réponse aux traitements en question. F n'a pas réussi à réduire l'accumulation de P, mais a pu réduire sa persistance dans les pousses de riz. D'autre part, P n'a pas réduit la persistance de F, mais a déterminé une accumulation plus que double de F dans les plantes. Donc a-t-on soulevé l'hypothèse d'un mécanisme de détoxication commun aux deux composés, mais favorable à P. Ce mécanisme consiste en une activation de l'activité GST à l'égard de P comme substrat [GST(P)] en réponse à tous les traitements. On a relevé que les niveaux d'activation étaient proportionnels à la quantité de P et F persistants, et additifs dans le cas du traitement avec le mélange P-F. Les paramètres cinétiques V max et K m de [GST(P)] ont été respectivement augmentés et diminués dans les pousses de riz traitées avec P, F et particulièrement avec le mélange P-F. On a eu donc confirmation d'un mécanisme de défense à l'égard de P, consistant en une expression accrue de [GST(P)] dans la plante de riz, en ce qui concerne tant la quantité globale de l'enzyme dans le «pull» protéique que l'induction des isoformes enzymatiques plus sensibles à P, en réponse à tous les traitements et surtout au traitement à base de mélange P-F. Ce résultat concorde avec ce qui a été observé quant à l'effet de F sur la persistance et la détoxication de P dans les pousses de riz. Oryza sativa / pretilachlore / fenclorim / persistence / glutathion-S-transférase