Mutant analysis in Arabidopsis provides insight into the molecular mode of action of the auxinic herbicide dicamba

Herbicides that mimic the natural auxin indole-3-acetic acid are widely used in weed control. One common auxin-like herbicide is dicamba, but despite its wide use, plant gene responses to dicamba have never been extensively studied. To further understand dicamba's mode of action, we utilized Arabidopsis auxin-insensitive mutants and compared their sensitivity to dicamba and the widely-studied auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The mutant axr4-2, which has disrupted auxin transport into cells, was resistant to 2,4-D but susceptible to dicamba. By comparing dicamba resistance in auxin signalling F-box receptor mutants (tir1-1, afb1, afb2, afb3, and afb5), only tir1-1 and afb5 were resistant to dicamba, and this resistance was additive in the double tir1-1/afb5 mutant. Interestingly, tir1-1 but not afb5 was resistant to 2,4-D. Whole genome analysis of dicamba-induced gene expression showed that 10 hours after application, dicamba stimulated many stress-responsive and signalling genes, including those involved in biosynthesis or signalling of auxin, ethylene, and abscisic acid (ABA), with TIR1 and AFB5 required for the dicamba-responsiveness of some genes. Research into dicamba-regulated gene expression and the selectivity of auxin receptors has provided molecular insight into dicamba-regulated signalling and could help in the development of novel herbicide resistance in crop plants.     

The chlorophenoxy herbicide dicamba and its commercial formulation banvel induce genotoxicity and cytotoxicity in Chinese hamster ovary (CHO) cells

The sister chromatid exchange (SCE) frequency, the cell-cycle progression analysis, and the single cell gel electrophoresis technique (SCGE, comet assay) were employed as genetic end-points to investigate the geno- and citotoxicity exerted by dicamba and one of its commercial formulation banvel (dicamba 57.71%) on Chinese hamster ovary (CHO) cells. Log-phase cells were treated with 1.0-500.0 microg/ml of the herbicides and harvested 24 h later for SCE and cell-cycle progression analyses. All concentrations assessed of both test compounds induced higher SCE frequencies over control values. SCEs increased in a non-dose-dependent manner neither for the pure compound (r=0.48; P>0.05) nor for the commercial formulation (r=0.58, P>0.05). For the 200.0 microg/ml and 500.0 microg/ml dicamba doses and the 500.0 microg/ml banvel dose, a significant delay in the cell-cycle progression was found. A regression test showed that the proliferation rate index decreased as a function of either the concentration of dicamba (r=-0.98, P<0.05) or banvel (r=-0.88, P<0.01) titrated into cultures in the 1.0-500.0 microg/ml dose-range. SCGE performed on CHO cells after a 90 min pulse-treatment of dicamba and banvel within a 50.0-500.0 microg/ml dose-range revealed a clear increase in dicamba-induced DNA damage as an enhancement of the proportion of slightly damaged and damaged cells for all concentrations used (P<0.01); concomitantly, a decrease of undamaged cells was found over control values (P<0.01). In banvel-treated cells, a similar overall result was registered. Dicamba induced a significant increase both in comet length and width over control values (P<0.01) regardless of its concentration whereas banvel induced the same effect only within 100.0-500.0 microg/ml dose range (P<0.01). As detected by three highly sensitive bioassays, the present results clearly showed the capability of dicamba and banvel to induce DNA and cellular damage on CHO cells.     

Crystal Structure of Dicamba Monooxygenase: A Rieske Nonheme Oxygenase that Catalyzes Oxidative Demethylation

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 Å resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.     

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: gene isolation, characterization, and heterologous expression

Dicamba O-demethylase is a multicomponent enzyme from Pseudomonas maltophilia, strain DI-6, that catalyzes the conversion of the widely used herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA (3,6-dichlorosalicylic acid). We recently described the biochemical characteristics of the three components of this enzyme (i.e. reductase(DIC), ferredoxin(DIC), and oxygenase(DIC)) and classified the oxygenase component of dicamba O-demethylase as a member of the Rieske non-heme iron family of oxygenases. In the current study, we used N-terminal and internal amino acid sequence information from the purified proteins to clone the genes that encode dicamba O-demethylase. Two reductase genes (ddmA1 and ddmA2) with predicted amino acid sequences of 408 and 409 residues were identified. The open reading frames encode 43.7- and 43.9-kDa proteins that are 99.3% identical to each other and homologous to members of the FAD-dependent pyridine nucleotide reductase family. The ferredoxin coding sequence (ddmB) specifies an 11.4-kDa protein composed of 105 residues with similarity to the adrenodoxin family of [2Fe-2S] bacterial ferredoxins. The oxygenase gene (ddmC) encodes a 37.3-kDa protein composed of 339 amino acids that is homologous to members of the Phthalate family of Rieske non-heme iron oxygenases that function as monooxygenases. Southern analysis localized the oxygenase gene to a megaplasmid in cells of P. maltophilia. Mixtures of the three highly purified recombinant dicamba O-demethylase components overexpressed in Escherichia coli converted dicamba to DCSA with an efficiency similar to that of the native enzyme, suggesting that all of the components required for optimal enzymatic activity have been identified. Computer modeling suggests that oxygenase(DIC) has strong similarities with the core alphasubunits of naphthalene 1,2-dioxygenase. Nonetheless, the present studies point to dicamba O-demethylase as an enzyme system with its own unique combination of characteristics.     

Effect of herbicide banvel on rabbit vaginal mucus membrane

Effect of banvel and its active ingredient, dicamba (3,6-dichloro-o-anisic acid) was investigated employing rabbit mucus membrane irritancy test. Inflammatory changes which did not exceed an average score of 2+ were observed in the animals 48 hr after a single intravaginal application of banvel (0.1 ml/rabbit) and dicamba (100 mg/rabbit). Persistent histopathological changes were observed in 1 out of 6 banvel-treated rabbits 15 days post-application. The results suggest that banvel and dicamba are not primary irritants but should nevertheless be employed with caution.     

Plant regeneration via somatic embryogenesis in creeping bentgrass (Agrostis palustris Huds.)

We have established a high-frequency plant regeneration system via somatic embryogenesis from mature seeds of creeping bentgrass (Agrostis palustris Huds). The effects of 2,4-dichlorophenoxyacetic acid (2,4-D), 3.6-dichloroo-anisic acid (dicamba) and 6-benzyladenine (BA) on callus formation and embryogenesis were evaluated. Callus produced on the Murashige and Skoog (MS) (1962) medium containing 2,4-D had low embryogenic potency. In the presence of 30 M dicamba, addition of 2.25 to 9 M BA significantly enhanced embryogenic callus formation over dicamba alone. Optimum frequency of somatic embryogenesis was achieved on MS basal medium containing 30 M dicamba and 2.25 M BA. Over 80% of somatic embryos germinated and formed plantlets on half-strength MS basal medium. These plantlets grew normally in the greenhouse.Abbreviations MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - dicamba 3, 6-dichloro-o-anisic acid     

Engineering dicamba selectivity in crops: a search for appropriate degradative enzyme(s)

The biotechnologial approaches to conferring crop selectivity to herbicides have been demonstrated for a number of compounds such as glyphosate, glufosinate, imidazolinones and cyclohexanediones. Imidazolinone-resistant and cyclohexanedione-resistant maize lines are already in the market. There are several other effective and environmentally benign herbicides such as dicamba, for which engineering crop selectivity is desirable, to broaden the product utility in different crops and provide new solutions for weed control. One of the most effective approaches to conferring dicamba selectivity in crops is to incorporate a gene for its rapid metabolism. It is advantageous to have different dicamba-metabolizing enzymes in order to maximize the chances of at least one functioning optimally in a plant environment. Three different metabolizing enzymes are currently available to engineer crop selectivity. The first one is the folate-dependent O-demethylase from Clostridium thermoaceticum, that converts dicamba to herbicidally inactive 3,6-dichlorosalicylate. The second enzyme is the NADH-dependent, multi-component monooxygenase from Pseudomonas maltophilia DI-6 that also converts dicamba to 3,6-dichlorosalicylate. The third enzyme is from corn endosperm cultures that catalyzes the 5-hydroxylation of dicamba. The merits of these three enzymes are discussed with respect to conferring crop selectivity to dicamba. In addition, a rapid microbial screen was conceived for discovery of new dicamba-degrading bacteria, which resulted in identification of Pseudomonas orvilla. This bacteria degraded dicamba by the same pathway, perhaps using a similar enzyme system as Pseudomonas maltophilia DI-6. However, the microbial screen has the potential to identify novel bacteria that degrade dicamba by a different pathway, providing more options for metabolizing enzymes to confer herbicide selectivity in crops. Received 13 February 1997/ Accepted in revised form 26 June 1997     

Comparative effects of herbicide dicamba and related compound on plant mitochondrial bioenergetics

The herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) was evaluated for its effects on bioenergetic activities of potato tuber mitochondria to elucidate putative mechanisms of action and to compare its toxicity with 2-chlorobenzoic acid. Dicamba (4 micro mol/mg mitochondrial protein) induces a limited stimulation of state 4 respiration of ca. 10%, and the above concentrations significantly inhibit respiration, whereas 2-chlorobenzoic acid maximally stimulates state 4 respiration (ca. 50%) at about 25 micro mol/mg mitochondrial protein. As opposed to these limited effects on state 4 respiration, transmembrane electrical potential is strongly decreased by dicamba and 2-chlorobenzoic acid. Dicamba (25 micro mol/mg mitochondrial protein) collapses, almost completely, Deltapsi; similar concentrations of 2-chlorobenzoic acid promote Deltapsi drops of about 50%. Proton permeabilization partially contributes to Deltapsi collapse since swelling in K-acetate medium is stimulated, with dicamba promoting a stronger stimulation. The Deltapsi decrease induced by dicamba is not exclusively the result of a stimulation on the proton leak through the mitochondrial inner membrane, since there was no correspondence between the Deltapsi decrease and the change on the O(2) consumption on state 4 respiration; on the contrary, for concentrations above 8 micro mol/mg mitochondrial protein a strong inhibition was observed. Both compounds inhibit the activity of respiratory complexes II and III but complex IV is not significantly affected. Complex I seems to be sensitive to these xenobiotics. In conclusion, dicamba is a stronger mitochondrial respiratory chain inhibitor and uncoupler as compared to 2-chlorobenzoic acid. Apparently, the differences in the lipophilicity are related to the different activities on mitochondrial bioenergetics.     

Effect of nitrogen and phosphorus status on the translocation of three herbicides in field bindweed (Convolvulus arvensis L.)

Twenty-eight day old field bindweed plants grown in culture solutions deficient in nitrogen (N) or phosphorus (P) for the last seven days of growth translocated significantly less foliarly applied dicamba (3,6-dichloro-o-anisic acid) and 2,4-D [(2,4-dichlorophenoxy) acetic acid] to their roots than did plants grown in complete nutrient solutions. In contrast, N deficiency stimulated basipetal translocation of glyphosate [N-(phosphonomethyl) glycine] and inhibited its acropetal translocation in field bindweed. Deficiencies of both N and P decreased translocation of dicamba from the treated area, but had no influence on translocation of glyphosate or 2,4-D from the treated area.Journal Article No. 4406 of the Agric. Exp. Stn., Oklahoma State University.     

麦草畏室内生测方法建立及大豆对麦草畏耐受性分析

室内生物测定是植物对除草剂等化学物质耐受性鉴定的一种常用筛选方法,已广泛应用于大豆、稗草、棉花等植物对草甘膦、氯嘧磺隆等除草剂的耐受性研究,但麦草畏的室内生物测定方法和大豆对麦草畏耐受性相关研究尚未见报道。本研究以麦草畏对催芽大豆下胚轴伸长抑制率为评价指标,结合回归方程曲线分析和抑制中浓度分析,建立了大豆对麦草畏耐受性室内生物测定方法,确定以300μg/L麦草畏筛选浓度作为大豆室内生物测定临界筛选浓度。利用该方法对35份源自微核心种质的大豆品种进行鉴定,结果表明,随麦草畏浓度增加,不同品种对麦草畏的耐受性存在显著差异,大豆品种对麦草畏的耐受性降低,从中筛选出对麦草畏耐受性较高的大黄豆-1和什邡螺丝豆。本研究结果为培育抗麦草畏品种的亲本选配以及后代选择提供了理论依据、材料和技术支撑。     

Differences among auxin treatments on haploid production in durum wheat × maize crosses

Three doubled haploid lines of durum wheat [Triticum turgidum ssp. durum (Desf.) Husn.] were crossed with maize (Zea mays L.), and five hormone treatments were applied to test their effect on the production of caryopses, embryos and haploid plants. The auxin treatments consisted of 100 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mg/l or 50 mg/l dicamba and two combination mixtures of 95/5 mg/l and 50/50 mg/l 2,4-D plus dicamba, respectively. Hormones were added to the culture medium of the detached tillers. Differences were not observed among the four hormone treatments that contained dicamba, nevertheless, these treatments significantly increased the production of caryopses, embryos and haploid plants. On average, 8.9 caryopses, 2.6 embryos and 1.3 haploid plants per spike were obtained following the treatment with 100 mg/l 2,4-D, and 15.0 caryopses, 6.0 embryos and 3.0 haploid plants per spike were obtained following the various treatments with dicamba. We propose the application of dicamba alone, or dicamba plus 2,4-D, as a means for improving the yield of haploid plants of durum wheat through crosses with maize.     

Somatic embryogenesis and plant regeneration in Sorghum bicolor (L.) Moench

Summary Callus induction, somatic embryogenesis and plant regeneration were obtained in two cultivars of Sorghum bicolor (L.) Moench. Transverse thin cell layers from roots/epicotyls of 15-day-old seedlings or of regenerated plantlets were used. Callus response depended on the genotype, the size of transverse thin cell layers, the level at which transverse thin cell layers were excised on the epicotyl, the composition of growth substances and the number of in vitro regeneration cycles undergone by the donor plant. Somatic embryos were differentiated under a defined dark/light sequence, from epidermised compact calluses (i.e having already differentiated an epidermis), obtained directly with dicamba or from friable callus initiated with kinetin and 2,4 dichlorophenoxyacetic acid. The importance of kinetin and dicamba on the induction of embryogenic potential is reported.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - 2iP N6-(2-isopentyl)adenine - BAP 6-benzylaminopurine - CaMV cauliflower mosaïc virus - CPPU N-(2-chloro 4-pyridyl)-N-phenylurea - dicamba 3,6-dichloro-o-anisic acid - IAA indole-3-acetic acid - K kinetin - MS Murashige and Skoog - NAA -naphthaleneacetic acid - PEPC phosphoenolpyruvate carboxylase - SD standard deviation - tTCL transverse thin cell layer     

Modeling Dicamba Sorption and Transport Through Zoysiagrass Thatch and Soil

The presence of turfgrass thatch complicates the sorption and transport of water soluble pesticides because the surface-applied pesticides must pass through an organic-rich thatch layer prior to entering the soil. The study was conducted (1) to determine the impact of zoysiagrass thatch (Zoy-sia japonica Steud.) on dicamba (3,6-dichloro-2-methoxy benzoic acid) transport through soil columns, and (2) to evaluate the effectiveness of linear equilibrium (LEM), two site nonequilibrium (2SNE) and one site nonequilibrium (1SNE) models to predict dicamba transport through columns containing a surface layer of thatch and columns devoid of thatch. The equilibrium sorption isotherms of ¹⁴C dicamba to homogenized samples of zoysiagrass thatch and a Sassafras loamy sand soil (fine loamy, mixed mesic, Typic Hapludult) were determined. Following the application of bromide to determine transport parameters, 0.56?kg dicamba ha^?1 was surface applied to undisturbed soil columns containing a surface layer of thatch and columns devoid of thatch and leachate samples collected for 12?h under steady-state unsaturated conditions. Zoysiagrass thatch (Kf = 0.82) had a three times greater sorption capacity than the soil (Kf = 0.28) beneath the thatch. Dicamba leaching for columns with thatch layers was ca. 21% less than soil columns devoid of thatch. When dicamba breakthrough curves were fitted to the different forms of the convective dispersive equation, the 2SNE model simulated dicamba transport better than LEM and 1SNE models, indicating the presence of two-site nonequilibrium sorption. Indications are that turfgrass thatch may have significant effects on dicamba leaching that presently used regulatory models based on LEM approach do not adequately consider.     

Plant regeneration from cocoyam callus derived from shoot tips and petioles

The effects of various growth regulators on morphogenesis from cocoyam tissues (Xanthosoma sagittifolium) were investigated. Calluses were initiated from shoot tip and petiole explants and proliferated on medium containing 1.36 μM dicamba. Callus production was significantly greater from petioles than from shoot tips. Thidiazuron (0.045 μM) enhanced callus production when dicamba (13.5 μM) was used, and was more favorable to petioles than shoot tips. Friable shoot tip callus was subcultured into liquid media containing either 1.36 μM dicamba alone, 1.35 μM 2,4-D + 0.46 μM kinetin or 1.36 μM dicamba + 0.46 μM kinetin to induce adventive regeneration. Tissues producing single or aggregated shoot buds were subcultured into media containing 0, 0.049 and 0.49 μM 2-isopentenyladenine where bud multiplication and shoot regeneration were observed. Bud aggregates were formed from callus in liquid cultures containing 1.36 μM dicamba, 1.36 μM dicamba + 0.46 μM kinetin or 1.35 μM 2,4-D + 0.46 μM kinetin. Shoot bud clumps which remained green produced shoots, daughter buds, and plantlets in stationary and agitated liquid media containing 0, 0.049 and 0.49 μM 2iP. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microbiological degradation of the herbicide dicamba

Summary Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba byP. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100–2000 mg dicamba L^–1 (0.5–9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl^– per mol of dicamba degraded. The cultures had an optimum pH 6.5–7.0 for dicamba degradation. Growth studies at 10°C, 20°C, and 30°C yielded activation energy values in the range of 19–36 kcal mol^–1 and an average Q₁₀ value of 4.0. Compared with the pure cultureP. paucimobilis, the consortium was more active at the lower temperature.     

Influence of dicamba and casein hydrolysate on somatic embryo number and culture quality in cell suspensions of Dactylis glomerata (Gramineae)

The effects of various concentrations and combinations of dicamba (3,6-dichloro-o-anisic acid) and casein hydrolysate on growth, mucilage accumulation, somatic embryo and root development in suspension cultures of Dactylis glomerata L. (orchardgrass) were examined. Fresh weight of culture tissue was increased with 20 M but not with 80 or 160 M dicamba in treatments with 1–4 g/l casein hydrolysate. Different casein hydrolysate concentrations did not alter the amount of mucilage (measured by viscosity) in the supernatant in the absence of dicamba. However, the addition of dicamba increased viscosity with 80 M giving the maximum response. Casein hydrolysate produced the greatest viscosity at 1–3 g/l in treatments where dicamba was present. Both dicamba and casein hydrolysate were required for development of somatic embryos. Dicamba at 40 M with 3–4 g/l casein hydrolysate produced approximately 2000 embryos/35 ml of suspension. Root development was inhibited by dicamba and stimulated by the presence of casein hydrolysate. The usefulness of medium component manipulations for influencing somatic embryogenesis and culture quality is discussed.     

Improvement of regeneration ability in Phleum pratense L. in vitro culture by dicamba

Calli were induced from mature caryopses of timothy grass (Phleum pratense L.) on MS medium (Murashige and Skoog 1962) supplemented with 500 mg·dm⁻³ casein hydrolysate and 5 mg·dm⁻³ 2,4-D (2,4-dicholorophenoxyacetic acid) or 2 mg·dm⁻³ dicamba (3,6-dichloro-o-anisic acid). Twelve-week-old calli were passaged on media with reduced levels of auxins (2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba). Tissues induced on medium with 2,4-D were transferred on medium with 2,4-D and on medium with dicamba; parallely calli initiated on medium with dicamba were passaged on medium with 2,4-D or dicamba. Calli from various media sequences were used to establish cell suspension cultures in media containing 2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba. An assessment of regeneration ability of calli was made on MS medium containing 0.2 mg·dm⁻³ kinetin. Callus tissue induced and/or subcultured on any of the media with 2,4-D did not regenerate plants while dicamba added to the media was the effective stimulator of regenerability. In the presence of 2,4-D calli and suspensions produced a jelly-like extracellular matrix. In cell suspension this phenomenon was observed 4–5 days after each passage. The measurements of electric potential of calli, growing on MS medium with kinetin were performed. Non-regenerating callus areas had an electric potential close to 0 mV while parts of tissue with meristematic centres were characterized by lower values of electric potential.     

Comparative Effects and Concentration of Picloram, 2,4,5-T and Dicamba in Tissue Culture

In nutrient agar comparative concentrations (10^?3 to 10^?5M) of (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T) were generally more inhibitory to the growth of tissue cultures of soybean (Glycine max (L.) Merrill cv. Acme) and cottonwood (Populus deltoides Marsh.) than were either 4-amino-3,5,6-trichloropicolinic acid (picloram) or 3,6-dichloro-o-anisic acid (dicamba). Compared to untreated tissue dicamba or picloram at 10^?6M in the nutrient agar resulted in a 200 % increase in the growth of soybean tissue. At 10^?5 and 10^?6M dicamba also produced an increase in the growth of cottonwood tissue. Greatest absorption of picloram and dicamba by tissue cultures from agar occurred during the first 24 h after treatment. However, absorption remained nearly static thereafter for 14 days. More dicamba was absorbed by soybean and cottonwood tissue cultures than either picloram or 2,4,5-T.     

Efficient plant regeneration system for immature embryos of triticale (x Triticosecale Wittmack)

A range of tissue culture conditions were tested to improve embryo culture frequency, and to develop an efficient plant regeneration system for triticale. Immature embryos (14–21 days post-anthesis) from two triticale genotypes (Hx87-139 and Tahara) were cultured on a commonly used Murashige and Skoog (MS) and on Lazzeri's (L1) basal medium with varied carbon sources, and two different plant growth regulators; 2,4-Dichlorophenoxyacetic acid (2,4-D) and 3,6-Dichloro-2-methoxybenzoic acid (dicamba). Although embryos could be cultured on both media types, L1 based medium was better than MS basal salts for callus induction and somatic embryogenesis, with plant regeneration frequencies up to 11 fold greater on L1 media types. In the presence of dicamba, callus induction was more rapid, that resulted in subsequent regeneration of up to 2 fold more plantlets than from callus induced on medium containing 2,4-D. Maltose appeared to be a superior carbon source during differentiation of callus. Genotype Tahara showed a better regenerative response than Hx87-138, with up to 23 normal, fertile plants being produced from a single embryo when cultured on L1MDic medium, containing maltose (5%) and dicamba (20 mg l^–1). Applications of this tissue culture procedure in triticale improvement through genetic engineering are also discussed.     

Comparative effects of the herbicides dicamba, 2,4-D and paraquat on non-green potato tuber calli

The effects of the herbicides 1,1'-dimethyl-4,4'-bipyridylium dichloride (paraquat), 3,6-dichloro-2-metoxybenzoic acid (dicamba) and 2,4-dichlorophenoxyacetic acid (2,4-D) on cell growth of non-green potato tuber calli are described. We attempted to relate the effects with toxicity, in particular the enzymes committed to the cellular antioxidant system. Cell cultures were exposed to the herbicides for a period of 4 weeks. Cellular integrity on the basis of fluorescein release was strongly affected by 2,4-D, followed by dicamba, and was not affected by paraquat. However, the three herbicides decreased the energy charge, with paraquat and 2,4-D being very efficient. Paraquat induced catalase (CAT) activity at low concentrations (1muM), whereas at higher concentrations, inhibition was observed. Dicamba and 2,4-D stimulated CAT as a function of concentration. Superoxide dismutase (SOD) activity was strongly stimulated by paraquat, whereas dicamba and 2,4-D were efficient only at higher concentrations. Glutathione reductase (GR) activity was induced by all the herbicides, suggesting that glutathione and glutathione-dependent enzymes are putatively involved in the detoxification of these herbicides. Paraquat slightly inhibited glutathione S-transferase (GST), whereas 2,4-D and dicamba promoted significant activation. These results indicate that the detoxifying mechanisms for 2,4-D and dicamba may be different from the mechanisms of paraquat detoxification. However, the main cause of cell death induced by paraquat and 2,4-D is putatively related with the cell energy charge decrease.