Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.     

Simultaneous assessment of the effects of an herbicide on the triad: rhizobacterial community, an herbicide degrading soil bacterium and their plant host

Aims

This work addresses the relevant effects that one single compound, used as model herbicide, provokes on the activity/survival of a suitable herbicide degrading model bacterium and on a plant that hosts this bacterium and its bacterial rhizospheric community.

Methods

The effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), on Acacia caven hosting the 2,4-D degrading bacterium Cupriavidus pinatubonensis JMP134, and its rhizospheric microbiota, were simultaneously addressed in plant soil microcosms, and followed by culture dependent and independent procedures, herbicide removal tests, bioprotection assays and use of encapsulated bacterial cells.

Results

The herbicide provokes deleterious effects on the plant, which are significantly diminished by the presence of the plant associated C. pinatubonensis, especially with encapsulated cells. This improvement correlated with increased 2,4-D degradation rates. The herbicide significantly changes the structure of the A. caven bacterial rhizospheric community; and it also diminishes the preference of C. pinatubonensis for the A. caven rhizosphere compared with the surrounding bulk soil.

Conclusions

The addition of an herbicide to soil triggers a complex, although more or less predictable, suite of effects on rhizobacterial communities, herbicide degrading bacteria and their plant hosts that should be taken into account in fundamental studies and design of bio(phyto)remediation procedures.     

Engineering 2,4-D resistance into cotton

Summary To reduce damage by drift-levels of the herbicide 2,4-dichlorophenoxyacetic acid, we have engineered the 2,4-D resistance trait into cotton (Gossypium hirsutum L.). The 2,4-D monooxygenase gene tfdA from Alcaligenes eutrophus plasmid pJP5 was isolated, modified and expressed in transgenic tobacco and cotton plants. Analyses of the transgenic progeny showed stable transmission of the chimeric tfdA gene and production of active 2,4-D monooxygenase. Cotton plants obtained were tolerant to 3 times the field level of 2,4-D used for wheat, corn, sorghum and pasture crops.     

Characterization of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid]-degrading Alcaligenes sp.CS1 and Ralstonia sp. CS2 isolated from agricultural soils

The herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] is widely applied to corn fields in order to control broad-leaved weeds. However, it is often detected in groundwater where it can be a persistent contaminant. Two mecoprop-degrading bacterial strains were isolated from agricultural soils through their capability to degrade ( R/S )-mecoprop rapidly. 16S rDNA sequencing of the isolates demonstrated that one was closely related to the genera Alcaligenes sp. (designated CS1) and the other to Ralstonia sp. (designated CS2). Additionally, these isolates demonstrated ability to grow on other related herbicides, including 2,4- D (2,4-dichlorophenoxyacetic acid), MCPA [4-chloro-2-methyl phenoxy acetic acid] and ( R/S )-2,4-DP [2-(2,4-dichlorophenoxy)propionic acid] as sole carbon sources. tfdABC gene-specific probes derived from the 2,4- D -degrading Variovorax paradoxus TV1 were used in hybridization analyses to establish whether tfd -like genes are present in mecoprop-degrading bacteria. Hybridization analysis demonstrated that both Alcaligenes sp. CS1 and Ralstonia sp. CS2 harboured tfdA , tfdB and tfdC genes on plasmids that have approximately > 60% sequence similarity to the tfdA , tfdB and tfdC genes of V. paradoxus . It is therefore likely that tfd -like genes may be involved in the degradation of mecoprop, and we are currently investigating this further.     

Transgenic tobacco plants overexpressing a cold-adaptive nitroreductase gene exhibited enhanced 2,4-dinitrotoluene detoxification rate at low temperature

Abstract

Plants encounter many environmental factors such as low and high temperatures during phytoremediation processes. In this study, our aim was to produce the transgenic tobacco plants by using a newly characterized bacterial nitroreductase, Ntr, which was active at a broad range temperature in order to detoxify 2,4-dinitrotoluene (2,4-DNT) at lower temperature. The presence of Ntr and its heterologous expression was verified in T1 transgenic plants and their growing ability were determined under toxic amount of 2,4-DNT (35?µM). Fresh weight and dry weight of transgenic plants were significantly higher than wild type (WT) under toxic 2,4-DNT at 22?°C, indicating higher growth capacity of the transgenics. Transgenic plants also showed a higher tolerance than WT when exposed to 2,4-DNT at 15?°C. Moreover, transformation rate of 2,4-DNT was gradually decreased through decreasing temperatures in WT media, however, it was increased through decreasing temperatures in transgenic plant TR3-25 media and it had the highest transformation rate (54%) of 2,4-DNT at 4?°C. Correlatively, 2,4-DNT treatment at 4?°C led to a significant decrease in H₂O₂ level in transgenic plants. Thus, transgenic plants overexpressing nitroreductase might have an important advantage for phytoremediation of toxic nitroaromatic compounds in field applications at low temperatures.     

Rhizobium leguminosarum bv. viciae 3841 Adapts to 2,4-Dichlorophenoxyacetic Acid with “Auxin-Like” Morphological Changes,Cell Envelope Remodeling and Upregulation of Central Metabolic Pathways

There is a growing need to characterize the effects of environmental stressors at the molecular level on model organisms with the ever increasing number and variety of anthropogenic chemical pollutants. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as one of the most widely applied pesticides in the world, is one such example. This herbicide is known to have non-targeted undesirable effects on humans, animals and soil microbes, but specific molecular targets at sublethal levels are unknown. In this study, we have used Rhizobium leguminosarum bv. viciae 3841 (Rlv) as a nitrogen fixing, beneficial model soil organism to characterize the effects of 2,4-D. Using metabolomics and advanced microscopy we determined specific target pathways in the Rlv metabolic network and consequent changes to its phenotype, surface ultrastructure, and physical properties during sublethal 2,4-D exposure. Auxin and 2,4-D, its structural analogue, showed common morphological changes in vitro which were similar to bacteroids isolated from plant nodules, implying that these changes are related to bacteroid differentiation required for nitrogen fixation. Rlv showed remarkable adaptation capabilities in response to the herbicide, with changes to integral pathways of cellular metabolism and the potential to assimilate 2,4-D with consequent changes to its physical and structural properties. This study identifies biomarkers of 2,4-D in Rlv and offers valuable insights into the mode-of-action of 2,4-D in soil bacteria.     

Effect of glucose on the amount of bacteria mineralizing 2,4-dichlorophenoxyacetic acid in soil

Plate numbers of bacteria and relative incidence of strains capable of mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in chernozem samples incubated for 14 d with the herbicide (50 ppm) in the presence or absence of glucose (1000 ppm) were compared. Whereas the total number of bacteria increased 1.2-fold in the variant with 2,4-D and 2.4-fold in the variant with glucose and the herbicide, the number of 2,4-D-mineralizing bacteria increased 12.1-fold and 34.2-fold, respectively. In a collection of 96 isolates of soil bacteria substantially more strains capable of degradation of 2,4-D in the presence of glucose were detected as compared with the variant without it, indicating that processes of cometabolic type are involved during the degradation of this herbicide in the soil.     

Differential response of young and adult leaves to herbicide 2,4-dichlorophenoxyacetic acid in pea plants: role of reactive oxygen species

In this work the differential response of adult and young leaves from pea (Pisum sativum L.) plants to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (23 mm) applied by foliar spraying was investigated. The concentration of 2,4-D (23 mm) and the time of treatment (72 h) were previously optimized in order to visualize its toxic effects on pea plants. Under these conditions, the herbicide induced severe disturbances in mesophyll cells structure and proliferation of vascular tissue in young leaves and increased acyl-CoA oxidase (ACX), xanthine oxidase (XOD) and lipoxygenase (LOX) activities in young leaves, and only ACX and LOX in adult leaves. This situation produced reactive oxygen species (ROS) over-accumulation favoured by the absence of significant changes in the enzymatic antioxidants, giving rise to oxidative damages to proteins and membrane lipids. An increase of ethylene took place in both young and adult leaves and the induction of genes encoding the stress proteins, PRP4A and HSP 71,2, was observed mainly in young leaves. These results suggest that ROS overproduction is a key factor in the effect of high concentrations of 2,4-D, and ROS can trigger a differential response in young and adult leaves, either epinasty development in young leaves or senescence processes in adult tissues.     

Cotton plants transformed with a bacterial degradation gene are protected from accidental spray drift damage by the herbicide 2,4-dichlorophenoxyacetic acid

The agronomic performance of broad leaved crop plants such as cotton would be greatly improved if genetically-engineered resistance to broadleaf herbicides could both protect the plants from accidental spray drift damage and allow the suppression of problem broadleaf weeds by chemical means. Followingin vitro modification and the addition of plant expression signals, the gene for 2,4-D monooxygenase, a bacterial enzyme that degrades the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), was introduced into cotton plants byAgrobacterium-mediated transformation. First generation homozygous progeny of regenerated transgenic cotton plants carrying this gene exhibited up to a 50–100 fold increase in tolerance to 2,4-D compared with untransformed controls, and glasshouse trials suggest that the genetically-engineered plants would be completely protected from spray drift of 2,4-D, at least up to the normal field application rates commonly used on neighbouring cereal crops.     

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.     

Role of a carboxylesterase in herbicide bioactivation in Arabidopsis thaliana

Arabidopsis thaliana contains multiple carboxyesterases (AtCXEs) with activities toward xenobiotics, including herbicide esters that are activated to their phytotoxic acids upon hydrolysis. On the basis of their susceptibility to inhibition by organophosphates, these AtCXEs are all serine hydrolases. Using a trifunctional probe bearing a fluorophosphonate together with biotin and rhodamine to facilitate detection and recovery, four dominant serine hydrolases were identified in the proteome of Arabidopsis. Using a combination of protein purification, capture with the trifunctional probe and proteomics, one of these hydrolases, AtCXE12, was shown to be the major carboxyesterase responsible for hydrolyzing the pro-herbicide methyl-2,4-dichlorophenoxyacetate (2,4-D-methyl) to the phytotoxic acid 2,4-dichlorophenoxyacetic acid. Recombinant expression of the other identified hydrolases showed that AtCXE12 was unique in hydrolyzing 2,4-D-methyl. To determine the importance of AtCXE12 in herbicide metabolism and efficacy, the respective tDNA knock-out (atcxe12) plants were characterized and shown to lack expression of AtCXE12 and have greatly reduced levels of 2,4-D-methyl-hydrolyzing activity. Young atcxe12 seedlings were less sensitive than wild type plants to 2,4-D-methyl, confirming a role for the enzyme in herbicide bioactivation in Arabidopsis.     

The broad-leaf herbicide 2,4-dichlorophenoxyacetic acid turns rice into a living trap for a major insect pest and a parasitic wasp

Synthetic chemical elicitors of plant defense have been touted as a powerful means for sustainable crop protection. Yet, they have never been successfully applied to control insect pests in the field. We developed a high-throughput chemical genetics screening system based on a herbivore-induced linalool synthase promoter fused to a β-glucuronidase (GUS) reporter construct to test synthetic compounds for their potential to induce rice defenses. We identified 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin homolog and widely used herbicide in monocotyledonous crops, as a potent elicitor of rice defenses. Low doses of 2,4-D induced a strong defensive reaction upstream of the jasmonic acid and ethylene pathways, resulting in a marked increase in trypsin proteinase inhibitor activity and volatile production. Induced plants were more resistant to the striped stem borer Chilo suppressalis, but became highly attractive to the brown planthopper Nilaparvata lugens and its main egg parasitoid Anagrus nilaparvatae. In a field experiment, 2,4-D application turned rice plants into living traps for N. lugens by attracting parasitoids. Our findings demonstrate the potential of auxin homologs as defensive signals and show the potential of the herbicide to turn rice into a selective catch crop for an economically important pest.     

A new concept for reduction of diffuse contamination by simultaneous application of pesticide and pesticide-degrading microorganisms

Pesticide residues and their transformation products are frequently found in groundwater and surface waters. This study examined whether adding pesticide-degrading microorganisms simultaneously with the pesticide at application could significantly reduce diffuse contamination from pesticide use. Degradation of the phenoxyacetic acid herbicides MCPA (4-chloro-2-methylphenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) was studied in soil microcosm experiments after simultaneous spraying of herbicide and herbicide-degrading bacteria on an agricultural soil and on a sand with low degradation potential. The latter represented pesticide use on non-agricultural soils poor in microbial activity. Degradation and possible loss of herbicidal effect were also tested in a system with plants and the amounts of bacteria needed to give satisfactory MCPA-degradation rate and the survival of degrading bacteria in formulated MCPA were determined. The results showed >80–99% degradation of 2,4-D and MCPA in soil within 1 day and >99% within 3 days after inoculation with 10⁵–10⁷ herbicide-degrading bacteria g⁻¹ dry weight of soil. Enhanced degradation of MCPA was also obtained in the presence of winter wheat and white mustard without loss of the intended herbicidal effect on white mustard. The survival of an isolated MCPA-degrading Sphingomonas sp. in three realistic concentrations of formulated MCPA was very poor, showing that in practical applications direct contact between the microorganisms and the pesticide formulation must be precluded. The applicability and economic feasibility of the method and the information needed to obtain a useable product for field use are discussed.     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

In vivo and in vitro binding of 2,4-dichlorophenoxyacetic acid to a rat liver mitochondrial protein

2,4-dichlorophenoxyacetic acid (2,4-D) is a hormonal herbicide widely used in the world because of its efficacy in the control of broadleaf and woody plants. In this study we have demonstrated in vivo covalent binding of the phenoxyherbicide 2,4-D to a single protein of 52 kD (from rat liver mitochondrial preparation) detected through immunoblotting studies with the specific antiserum for 2,4-D. The direct involvement of 2,4-D in the formation of the adduct has also been demonstrated in vitro, using liver mitochondrial preparations exposed to 14C-UL-2,4-D. Radiolabeled protein separated by SDS-PAGE and afterwards electroeluted showed a single labeled protein of 52 kD. When mitochondria exposed to radiolabeled xenobiotic were devoid of their outer membrane, the specific activity observed suggest that protein involved in covalent interaction belongs to the inner mitochondrial membrane. We propose that covalent binding of the phenoxyherbicide 2,4-D to a very specific single protein of 52 kD observed in vitro and in vivo may be related to known alterations of the mitochondrial function.     

Quantitative distribution and metabolism of auxin herbicides in roots

The internal concentrations of four auxin herbicides— 2,4-dichlorophenoxyacetic acid, dicamba, picloram, and naphthaleneacetic acid—were measured in the roots of treated pea seedlings. Intact seedlings were immersed in solutions of labeled herbicides at concentrations sufficient to produce toxic symptoms (inhibition of elongation, radial enlargement, and lateral root proliferation). Measurements of volume and herbicide content of segments taken sequentially along the root showed that an acropetal concentration gradient of each herbicide was established within the root immediately following treatment. Although there was a net loss of herbicide in the following 24 hours, the gradient was maintained. Initially, the concentration of herbicide in the root tips exceeded that in the external medium.     

rRNA operon copy number reflects ecological strategies of bacteria

Although natural selection appears to favor the elimination of gene redundancy in prokaryotes, multiple copies of each rRNA-encoding gene are common on bacterial chromosomes. Despite this conspicuous deviation from single-copy genes, no phenotype has been consistently associated with rRNA gene copy number. We found that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond to resource availability. Soil bacteria that formed colonies rapidly upon exposure to a nutritionally complex medium contained an average of 5.5 copies of the small subunit rRNA gene, whereas bacteria that responded slowly contained an average of 1.4 copies. In soil microcosms pulsed with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), indigenous populations of 2,4-D-degrading bacteria with multiple rRNA genes ( = 5.4) became dominant, whereas populations with fewer rRNA genes ( = 2.7) were favored in unamended controls. These findings demonstrate phenotypic effects associated with rRNA gene copy number that are indicative of ecological strategies influencing the structure of natural microbial communities.     

Isolation and characterization of a stable 2,4-dichlorophenoxyacetic acid degrading bacterium, Variovorax paradoxus, using chemostat culture

A strain of Variovorax paradoxus degrading 2,4-dichlorophenoxyacetic acid (2,4-D) was isolated from the Dijon area (France) using continuous chemostat culture. This strain, designated TV1, grew on up to 5 mM 2,4-D and efficiently degraded the herbicide as sole carbon source as well as in presence of soil extracts. It also degraded phenol and 2-methyl, 4-chlorophenoxyacetic acid at 3 mM and 2,4-dichlorophenol at 1 mM. This organism contained a stable 200 kb plasmid, designated pTV1, which showed no similarity in its restriction pattern with the archetypal 2,4-D catabolic plasmid pJP4. However, pTV1 contained an 11 kb BamHI fragment which hybridized at low stringency with the 2,4-D degradative genes tfdA, tfdB and tfdR from pJP4. PTV1 partial tfdA sequence showed 77 % similarity with the archetypal tfdA gene sequence from Ralstonia eutropha JMP134. Tn5 mutagenesis confirmed the involvement of this gene in the 2,4-D catabolic pathway. © Rapid Science Ltd. 1998