Atrazine metabolism and herbicidal selectivity

Metabolism of the herbicide 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) was investigated in resistant corn (Zea mays L.) and sorghum (Sorghum vulgare Pers.), intermediately susceptible pea (Pisum sativum L.), and highly susceptible wheat (Triticum vulgare Vill.) and soybean (Glycine max Merril.). This study revealed that 2 possible pathways for atrazine metabolism exist in higher plants. All species studied were able to metabolize atrazine initially by N-dealkylation of either of the 2 substituted alkylamine groups. Corn and wheat, which contain benzoxazinone, also metabolized atrazine initially by hydrolysis in the 2-position of the s-triazine ring to form hydroxyatrazine. Subsequent metabolism by both pathways resulted in the conversion of the parent atrazine to more polar compounds and eventually into methanol-insoluble plant residue. No evidence for s-triazine ring cleavage was obtained.

Both pathways for atrazine metabolism appear to detoxify atrazine. The hydroxylation pathway results in a direct conversion of a highly phytotoxic compound to a completely non-phytotoxic derivative. The dealkylation pathway leads to detoxication through one or more partially detoxified, stable intermediates. Therefore, the rate and pathways of atrazine metabolism are important in determining the tolerance of plants to the herbicide. Both quantitative and qualitative differences in atrazine metabolism were detected between resistant, intermediately susceptible, and susceptible species. The ability of plants to metabolize atrazine by N-dealkylation and the influence of this pathway in determining tolerance of plants to atrazine are discussed.

     

Glutathione conjugation: atrazine detoxication mechanism in corn

Glutathione conjugation (GS-atrazine) of the herbicide, 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) is another major detoxication mechanism in leaf tissue of corn (Zea mays, L.). The identification of GS-atrazine is the first example of glutathione conjugation as a biotransformation mechanism of a pesticide in plants. Recovery of atrazine-inhibited photosynthesis was accompanied by a rapid conversion of atrazine to GS-atrazine when the herbicide was introduced directly into leaf tissue. N-De-alkylation pathway is relatively inactive in both roots and shoots. The nonenzymatic detoxication of atrazine to hydroxyatrazine is negligible in leaf tissue. The hydroxylation pathway contributed significantly to the total detoxication of atrazine only when the herbicide was introduced into the plant through the roots. The metabolism of atrazine to GS-atrazine may be the primary factor in the resistance of corn to atrazine.     

EFFECTS OF S-TRIAZINES ON PROTEIN AND FINE STRUCTURE OF COTYLEDONS OF BUSH BEANS

A significant increase in protein content of bean cotyledons resulted by applications of 0.5 ppm of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine), simazine [2-chloro-4,6-bis(ethylamino)-s-triazine], terbutryn (2-methylmercapto-4-ethylamino-6-isobutylamino-.s-triazine), or GS-14254 (2-methoxy-4-isopropylamino-6-butylamino-s-triazine) to the foliage of 5-6 week old bean plants grown in a controlled environment or field conditions. Aen electron microscopic study indicated that in the cotyledonary cells s-triazines inducd a 2-fold increase in the number of cisternae of rough endoplasmic reticulum. These treatments also increased the number of vesicles, which apparently contain protein, and the amount of cytoplasmic ribosomes.     

Enzymatic Degradation of Chlorodiamino-s-Triazine

2-Chloro-4,6-diamino-s-triazine (CAAT) is a metabolite of atrazine biodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzes the dechlorination of atrazine but is unreactive with CAAT. In this study, melamine deaminase (TriA), which is 98% identical to AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-s-triazine (CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase (AtzB) to yield ammelide. This represents a newly discovered dechlorination reaction for AtzB. Ammelide was subsequently hydrolyzed by N-isopropylammelide isopropylaminohydrolase to produce cyanuric acid, a compound metabolized by a variety of soil bacteria.     

4-Amino-2-chloro-1,3,5-triazine: A New Metabolite of Atrazine by a Soil Bacterium

The degradation of herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1, 3, 5-triazine) by a soil bacterium is reported. The bacterium involved is a species of Nocardia, which utilizes the atrazine as the sole source of carbon and nitrogen. A new metabolite, 4-amino-2-chloro-1, 3, 5-triazine, of the degradation of atrazine in the presence of glucose has been identified. The results further substantiated that atrazine can be degraded by soil microorganisms and indicated that deamination can also occur, as well as dealkylation. 4-Amino-2-chloro-1,3,5-triazine did not show phytotoxic activity to oat (Avena sativa L.), demonstrating that deamination insures detoxification.     

Effects of Photosystem II Herbicides on the Photosynthetic Membranes of the Cyanobacterium Aphanocapsa 6308

The effects of the photosystem II herbicides diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on the photosynthetic membranes of a cyanobacterium, Aphanocapsa 6308, were compared to the effects on a higher plant, Spinacia oleracea. The inhibition of photosystem II electron transport by these herbicides was investigated by measuring the photoreduction of the dye 2,6-dichlorophenol-indophenol spectrophotometrically using isolated membranes. The concentration of herbicide that caused 50% inhibition of electron transport (I₅₀ value) in Aphanocapsa membranes for diuron was 6.8 × 10⁻⁹ molar and the I₅₀ value for atrazine was 8.8 × 10⁻⁸ molar. ¹⁴C-labeled diuron and atrazine were used to investigate herbicide binding with calculated binding constants (K) being 8.2 × 10⁻⁸ molar for atrazine and 1.7 × 10⁻⁷ molar for diuron. Competitive binding studies carried out on Aphanocapsa membranes using radiolabeled [¹⁴C]atrazine and unlabeled diuron revealed that diuron competed with atrazine for the herbicide-binding site. Experiments involving the photoaffinity label [¹⁴C]azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-2-triazine) and autoradiography of polyacrylamide gels indicated that the herbicide atrazine binds to a 32-kilodalton protein in Aphanocapsa 6308 cell extracts.     

Influence of s-Triazines on Some Enzymes of Carbohydrates and Nitrogen Metabolism in Leaves of Pea (Pisum sativum L.) and Sweet Corn (Zea mays L.)

Foliar applications of 2 milligrams per liter of 2-chloro-4,6-bis (ethylamino)-s-triazine, 2-methylmercapto-4-ethylamino-6-isobutylamino-s-triazine, and 2-methoxy-4-isopropylamino-6-butylamino-s-triazine caused increases in the activities of starch phosphorylase, pyruvate kinase, cytochrome oxidase, and glutamate dehydrogenase 5, 10, and 15 days after treatment in the leaves of 3-week-old seedlings of pea (Pisum sativum L.) and sweet corn (Zea mays L.). The results indicate that sublethal concentrations of s-triazine compounds affect the physiological and biochemical events in plants which favor more utilization of carbohydrates for nitrate reduction and synthesis of amino acids and proteins.     

Isolation and Characterization of a Novel Atrazine Metabolite Produced by the Fungus Pleurotus pulmonarius, 2-Chloro-4-Ethylamino-6-(1-Hydroxyisopropyl)Amino-1,3,5-Triazine

The white rot fungus Pleurotus pulmonarius exhibited metabolism of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in liquid culture, producing the dealkylated metabolites desethylatrazine, desisopropylatrazine, and desethyl-desisopropylatrazine. A fourth, unknown metabolite was also produced. It was isolated and was identified as 2-chloro-4-ethylamino-6-(1-hydroxyisopropyl)amino-1,3,5-triazine by gas chromatography-mass spectrometry, Fourier transformed infrared spectroscopy, and ¹H nuclear magnetic resonance analysis. The structure of this metabolite was confirmed by chemical synthesis of the compound and comparison with the fungally produced metabolite.     

Influence of S-ethyl Dipropylthiocarbamate on Atrazine Absorption by Wheat

Wheat (Triticum sativum L. cv. Nisu) grown in 0·5 Hoaglandssolution containing sub-toxic concentrations of S-ethyl dipropylthiocarbamate(EPTQ (0,0·0625,0·125,0·25, and 0·5p.p.m.w.) were exposed to ¹⁴C-ring labelled-2-chloro-4-ethylamino-6-isopropylamino-s-triazine(atrazine). Total ¹⁴C-atrazine absorption was increased to 182per cent in wheat treated with 0•5 p.p.m.w. EPTC when comparedto the EPTC untreated wheat. Detoxification and metabolism ofEPTC were not appreciably altered by EPTC pretreatment. Thisresulted in an increased atrazine content in the wheat leavespretreated with 0·5 p.p.m.w. EPTC that amounted to 370per cent of the unchanged atrazine present in the leaves ofEPTC untreated wheat.     

Biochemical and biophysical characterization of herbicide-resistant mutants of the unicellular cyanobacterium,Anacystis nidulans R2

Two herbicide-resistant mutants of the unicellular cyanobacterium, Anacystis nidulans R2, were obtained by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. These mutants, A. nidulans R2D1 and R2D2, were selected by growth of mutagenized cells in the presence of 10^?6 M and 10^?5 M 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), respectively. Both were found to be cross-resistant to 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) and 2-n-heptyl-4-hydroxyquinoline-n-oxide (HQNO) by measurement of Photosystem II activity in the presence of the inhibitors. The DCMU-resistance trait from each mutant was transferred to a wild-type genetic background by DNA-mediated transformation of A. nidulans cells. The two resulting transformants, A. nidulans R2D1-X1 and R2D2-X1, were similar to the original mutants with respect to DCMU- and HQNO-resistance. However, both exhibited increased sensitivity to atrazine relative to the mutants from which they were derived. Polyacrylamide gel electrophoretic analysis revealed that the mutants and transformants were deficient in a 34 kDa, surface-exposed polypeptide which was present in the wild-type strain; the transformants exhibited a new polypeptide of 35.5 kDa which was also highly surface-exposed.     

Study of soil algae

Summary The application of the algal method for soil bioassay has been extended to herbicide (atrazine). The method is capable of detecting less than 0.5 ppm of atrazine in solution. In residual herbicide assay the method is suitable for screening soils. For herbicide requirement prediction, the technique appeared to be superior to chemical analysis and applicable to soils of widely different characteristics. This has been demonstrated with oat pot trial.-2-chloro-4-ethylamino-6 isopropylamino-s-triazine.     

Purification and Characterization of an Inducible s-Triazine Hydrolase from Rhodococcus corallinus NRRL B-15444R

The widespread use and relative persistence of s-triazine compounds such as atrazine and simazine have led to increasing concern about environmental contamination by these compounds. Few microbial isolates capable of transforming substituted s-triazines have been identified. Rhodococcus corallinus NRRL B-15444 has previously been shown to possess a hydrolase activity that is responsible for the dechlorination of the triazine compounds deethylsimazine (6-chloro-N-ethyl-1,3,5-triazine-2,4-diamine) (CEAT) and deethylatrazine (6-chloro-N-isopropyl-1,3,5-triazine-2,4-diamine) (CIAT). The enzyme responsible for this activity was purified and shown to be composed of four identical subunits of 54,000 Da. Kinetic experiments revealed that the purified enzyme is also capable of deaminating the structurally related s-triazine compounds melamine (2,4,6-triamino-1,3,5-triazine) (AAAT) and CAAT (2-chloro-4,6-diamino-1,3,5-triazine), as well as the pyrimidine compounds 2,4,6-triaminopyrimidine (AAAP) and 4-chloro-2,6-diaminopyrimidine (CAAP). The triazine herbicides atrazine and simazine inhibit the hydrolytic activities of the enzyme but are not substrates. Induction experiments demonstrate that triazine hydrolytic activity is inducible and that this activity rises approximately 20-fold during induction.     

Enzymatic degradation of chlorodiamino-s-triazine

2-Chloro-4,6-diamino-s-triazine (CAAT) is a metabolite of atrazine biodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzes the dechlorination of atrazine but is unreactive with CAAT. In this study, melamine deaminase (TriA), which is 98% identical to AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-s-triazine (CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase (AtzB) to yield ammelide. This represents a newly discovered dechlorination reaction for AtzB. Ammelide was subsequently hydrolyzed by N-isopropylammelide isopropylaminohydrolase to produce cyanuric acid, a compound metabolized by a variety of soil bacteria.     

Evolution of atrazine-degrading capabilities in the environment

Since their first introduction in the mid 1950s, man-made s-triazine herbicides such as atrazine have extensively been used in agriculture to control broadleaf weed growth in different crops, and thus contributed to improving crop yield and quality. Atrazine is the most widely used s-triazine herbicide for the control of weeds in crops such as corn and sorghum. Although atrazine was initially found to be slowly and partially biodegradable, predominantly by nonspecific P450 monoxygenases which do not sustain microbial growth, microorganisms gradually evolved as a result of repeated exposure, started using it as a growth substrate and eventually succeeded in mineralizing it. Within three decades, an entirely new hydrolase-dependent pathway for atrazine mineralization emerged and rapidly spread worldwide among genetically different bacteria. This review focuses on the enzymes involved in atrazine mineralization and their evolutionary histories, the genetic composition of microbial populations involved in atrazine degradation and the biotechnologies that have been developed, based on these systems, for the bioremediation of atrazine contamination in the environment.     

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein.The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the D1 polypeptide, as shown with PS II membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.Abbreviations atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine - Chl chlorophyll, diuron - (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DPC diphenylcarbazide - ioxynil 4-cyano-2,6-diiodophenol - k_b binding constant - Mes 4-morpholinoethanesulfonic acid - P-680 reaction-center chlorophyll a of photosystem-II - PAGE polyacrylamide gel electrophoresis - PS II photosystem-II - Q_A and Q_B primary and secondary quinone electron acceptors - Z electron donor to the photosystem-II reaction center - SDS sodium dodecylsulfate - Tricine N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine     

Influence of some herbicides on the reproduction of tobacco mosaic virus inNicotiana tabacum cv. ‘Samsun’

The application of 10^?3 g 1^?1 of 2,4 dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, 3-amino-1,2,4-triazole and 6-chloro-2-ethylamino-4-isopropyl-amino-1,3,5-triazine toNicotiana tabacum cv. ‘Samsun’ plants inoculated with tobacco mosaic virus results in an increase in the content of this virus in the tissues. When whole plants are used, TMV content increases by 20% after herbicide application; when leaf dises are used, the amount of the virus can be doubled by the herbicide in comparison with control untreated inoculated discs. The results clearly show that the used non-selective herbicides act as stimulators of virus biosynthesis, probably via enhanced pentose phosphate cycle activity which enables an enhanced formation of viral RNA.     

Apoplastic and symplastic pathways of atrazine and glyphosate transport in shoots of seedling sunflower

[¹⁴C]Atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) and [¹⁴C]glyphosate (N-[phosphonomethyl]glycine) were xylem fed to sunflower shoots at 100 micromolar for 1 hour in the light, then placed in the dark at 100% relative humidity for 1, 4, 7, or 10 hours. The distribution of atrazine and glyphosate between shoot parts, in the leaves, and between the aoplast and symplast of the leaf was determined. The apoplastic concentrations and distribution patterns of atrazine and glyphosate in the leaves were evaluated using a pressure dehydration technique, our results were compared to the previously reported distribution patterns of the naturally occurring apoplastic leaf solutes, and the apoplastic dye PTS (trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate). The pattern of atrazine and glyphosate distribution in the shoot, and between the leaf apoplast and symplast, was found to reflect the potential of these herbicides to enter the shoot symplast. The results of this study are discussed with respect to current theories of xenobiotic transport in plants, and have been found to be consistent with the intermediate permeability hypothesis for xenobiotic transport.     

On the mutagenic and recombinogenic activity of certain herbicides in Salmonella typhimurium and in Aspergillus nidulans

The plant growth-regulating hormones indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), both strong recombinogens in Aspergillus nidulans, were tested in Salmonella typhimurium strains for his revertants at a range of concentrations from 1 to 2000 micrograms/plate with and without metabolic activation and were found negative. Also 3 herbicides of the chlorophenoxy group, 2,4-(dichlorophenoxy)acetic acid (2,4-D), 2,4-(dichlorophenoxy)butyric acid (2,4-DB) and 4-chloro-2-methylphenoxyacetic acid (MCPA), which show a plant growth hormone-like activity, and 2 of the triazine group, 2-ethylamino-4-chloro-6-isopropylamino-1,3,5-triazine (atrazine) and 2,4-bis(isopropylamino)6-chloro-1,3,5-triazine (propazine) were tested in S. typhimurium for point mutations and in A. nidulans for mitotic recombination. 2,4-D and MCPA were found to be weakly mutagenic at concentrations between 250 and 750 micrograms/plate in strain TA97a and only after metabolic activation and were recombinogens by inducing mainly mitotic crossing-over in A. nidulans at concentrations of 4-48 microM and 1500-3000 microM, respectively. 2,4-DB, atrazine and propazine were negative in both the Ames and the Aspergillus tests.     

Characterization of S-Triazine Herbicide Metabolism by a Nocardioides sp. Isolated from Agricultural Soils

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Nine gram-positive bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from four farms in central Canada. The strains were divided into two groups based on repetitive extragenic palindromic (rep)-PCR genomic fingerprinting with ERIC and BOXA1R primers. Based on 16S ribosomal DNA sequence analysis, both groups were identified as Nocardioides sp. strains. None of the isolates mineralized [ring-U-¹⁴C]atrazine. There was no hybridization to genomic DNA from these strains using atzABC cloned from Pseudomonas sp. strain ADP or trzA cloned from Rhodococcus corallinus. S-Triazine degradation was studied in detail in Nocardioides sp. strain C190. Oxygen was not required for atrazine degradation by whole cells or cell extracts. Based on high-pressure liquid chromatography and mass spectrometric analyses of products formed from atrazine in incubations of whole cells with H₂¹⁸O, sequential hydrolytic reactions converted atrazine to hydroxyatrazine and then to the end product N-ethylammelide. Isopropylamine, the putative product of the second hydrolytic reaction, supported growth as the sole carbon and nitrogen source. The triazine hydrolase from strain C190 was isolated and purified and found to have a K_m for atrazine of 25 μM and a V_max of 31 μmol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 μM EDTA but was inhibited by 100 μM Mg, Cu, Co, or Zn. Whole cells and purified triazine hydrolase converted a range of chlorine or methylthio-substituted herbicides to the corresponding hydroxy derivatives. In summary, an atrazine-metabolizing Nocardioides sp. widely distributed in agricultural soils degrades a range of s-triazine herbicides by means of a novel s-triazine hydrolase.     

Mineralization of the s-triazine ring of atrazine by stable bacterial mixed cultures.

Enrichment cultures containing atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) at a concentration of 100 ppm (0.46 mM) as a sole nitrogen source were obtained from soils exposed to repeated spills of atrazine, alachlor, and metolachlor. Bacterial growth occurred concomitantly with formation of metabolites from atrazine and subsequent biosynthesis of protein. When ring-labeled [14C]atrazine was used, 80% or more of the s-triazine ring carbon atoms were liberated as 14CO2. Hydroxyatrazine may be an intermediate in the atrazine mineralization pathway. More than 200 pure cultures isolated from the enrichment cultures failed to utilize atrazine as a nitrogen source. Mixing pure cultures restored atrazine-mineralizing activity. Repeated transfer of the mixed cultures led to increased rates of atrazine metabolism. The rate of atrazine degradation, even at the elevated concentrations used, far exceeded the rates previously reported in soils, waters, and mixed and pure cultures of bacteria.