Phytodegradation of organophosphorus compounds by transgenic plants expressing a bacterial organophosphorus hydrolase

Organophosphorus (OP) compounds are widely used as pesticides in agriculture but cause broad-area environmental pollution. In this work, we have expressed a bacterial organophosphorus hydrolase (OPH) gene in tobacco plants. An assay of enzyme activity showed that transgenic plants could secrete OPH into the growth medium. The transgenic plants were resistant to methyl parathion (Mep), an OP pesticide, as evidenced by a toxicity test showing that the transgenic plants produced greater shoot and root biomass than did the wild-type plants. Furthermore, at 0.02% (v/v) Mep, the transgenic plants degraded more than 99% of Mep after 14 days of growth. Our work indicates that transgenic plants expressing an OPH gene may provide a new strategy for decontaminating OP pollutants.     

Hydrolysis of organophosphorus compounds by microbial enzymes

There are classes of microbial enzymes that have the ability to degrade harmful organophosphorus (OP) compounds that are present in some pesticides and nerve agents. To date, the most studied and potentially important OP-degrading enzymes are organophosphorus hydrolase (OPH) and organophosphorus acid anhydrolase (OPAA), which have both been characterized from a number of organisms. Here we provide an update of what is experimentally known about OPH and OPAA to include their structures, substrate specificity, and catalytic properties. Current and future potential applications of these enzymes in the hydrolysis of OP compounds are also addressed.     

The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5–6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.     

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes were investigated in seeds and in seedlings and calli cultures ofC. peruvianus to determine if the changes in MDH isozyme banding patterns could be used as biochemical markers to identify the origin of regenerated plants from callus tissues. Four cytoplasmic MDH isozymes (sMDH), five mitochondrial MDH isozymes (mMDH), and one glyoxysomal MDH isozyme (gMDH) were detected and showed tissue- and stage-specific expression. A relationship of mMDH and gMDH isozyme patterns with callus tissues subcultured in three hormonal combinations and with the plants regenerated from these callus tissues was demonstrated. Furthermore, temperature and mechanical stress were found to be closely related to mMDH-1 activity in callus culture. Therefore, the different patterns of MDH isozymes in the various tissues ofC. peruvianus can be used as biochemical markers for the study of gene expression during development and as powerful tools in monitoring studies on callus cultures. This research was supported by the CNPq.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench) using an improved in vitro regeneration system

An improved regeneration protocol suitable for transformation of sorghum was developed. The improvements focused on limiting the production of phenolic compounds and the use of suitable culture vessels for each developmental stage in plant regeneration from immature embryo derived calli. The addition of activated charcoal in the callus induction medium reduced the production of black pigments, however it also inhibited the callus formation on immature embryo explants. Cold pre-treatment of the immature seeds from which embryo explants were excised had a positive effect on both explant survival and callus formation. A one-day 4°C treatment of immature seeds significantly improved the callus formation from immature embryos and reduced the need for frequent subculture. Petri dishes with ventilation were suitable for the callus induction phase, but not for plant regeneration. Regeneration of plants could be improved by using disposal plastic boxes (250 ml volume) instead of Petri dishes. Agrobacterium-mediated transformation using the improved regeneration protocol and the hygromycin phosphotransferase gene as selectable marker resulted in the recovery of 15 transgenic plants from 300 initial immature embryos (5% efficiency). The transgenic nature of the obtained plants was demonstrated by Southern hybridisation and progeny analysis. The transgenes were inherited in a Mendelian fashion and were integrated at a single locus in the majority of the analysed lines.     

Detoxification of the organophosphate nerve agent coumaphos using organophosphorus hydrolase immobilized on cellulose materials

Neurotoxic organophosphates (OPs) are widely used as pesticides and for public health purposes, as well as being nerve gases. As a result of the widespread use of these compounds for agriculture, large volumes of wastewater are generated. Additionally, there are large stockpiles of the nerve gases soman, sarin and VX in the United States and elsewhere around the world. Organophosphorus hydrolase (OPH) is an enzyme that catalyzes the hydrolysis of OP nerve agents. To date, however, the use of this enzyme in detoxification processes has been rather limited due to the high cost of its purification and short catalytic half-life. This paper reports the development of a cost-effective method for the production and immobilization of OPH in a pilot application in an enzyme bioreactor column for detoxification of paraoxon and coumaphos in contaminated wastewaters. A fusion between OPH and a cellulose binding domain that binds selectively to cellulose was generated to allow one-step purification and immobilization of OPH on cheap and abundantly available cellulose immobilization matrices. When packed in a column bioreactor, the immobilized fusion enzyme was able to completely degrade coumaphos up to a concentration of 0.2 mM. However, stirring of OPH immobilized on cellulose materials resulted in complete OP degradation of 1.5 mM coumaphos. The bioreactor column degraded the compounds tested at high concentration, rapidly, and without loss of process productivity for about 2 months.     

Altering the substrate specificity of organophosphorus hydrolase for enhanced hydrolysis of chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k(cat)/K(m) value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

RAPD markers to evaluate callus tissue of Cereus peruvianus Mill. (Cactaceae) maintained in different growth regulator combinations

RAPD markers were used to detect DNA polymorphisms in callus tissues maintained at different auxin and cytokinin combinations. There is a higher level of genetic variablity in callus tissue maintained with the highest kinetin versus 2, 4-D concentration. Callus tissues subcultured in a 4.0 mg/L 2,4-D and 4.0 mg/L kinetin combination showed high similarity and can be recommended as more suitable sources for industrial procedures of extraction of natural products such as secondary metabolites since extraction protocols can be easily standardized using genetically uniform materials. The higher genetic diversity in callus tissues of C. peruvianus cultured at 4.0 mg/L 2,4-D and 8.0 mg/L kinetin indicates this tissue as a matrix for in vitro selection of cell lines for higher natural products production. RAPD markers are, therefore, effective tools useful for detecting DNA polymorphism in callus tissue as well as in the DNA identification of callus tissues maintained in different auxin and cytokinin combinations.     

Immobilization of organophosphate hydrolase on biocompatible gelatin pads and its use in removal of organophosphate compounds and nerve agents

Bacterial organophosphate hydrolases (OPH) have been shown to hydrolyze structurally diverse group of organophosphate (OP) compounds and nerve agents. Due to broad substrate range and unusual catalytic properties, the OPH has successfully been used to develop eco-friendly strategies for detection and decontamination of OP compounds. However, their usage has failed to gain necessary acceptance, due to short half-life of the enzyme and loss of activity during process development. In the present study, we report a simple procedure for immobilization of OPH on biocompatible gelatin pads. The covalent coupling of OPH using glutaraldehyde spacer has been found to dramatically improve the enzyme stability. There is no apparent loss of OPH activity in OPH-gelatin pads stored at room temperature for more than six months. As revealed by a number of kinetic parameters, the catalytic properties of immobilized enzyme are found to be comparable to the free enzyme. Further, the OPH-gelatin pads effectively eliminate OP insecticide methyl parathion and nerve agent sarin.     

Detoxification of the organophosphate nerve agent coumaphos using organophosphorus hydrolase immobilized on cellulose materials

Neurotoxic organophosphates (OPs) are widely used as pesticides and for public health purposes, as well as being nerve gases. As a result of the widespread use of these compounds for agriculture, large volumes of wastewater are generated. Additionally, there are large stockpiles of the nerve gases soman, sarin and VX in the United States and elsewhere around the world. Organophosphorus hydrolase (OPH) is an enzyme that catalyzes the hydrolysis of OP nerve agents. To date, however, the use of this enzyme in detoxification processes has been rather limited due to the high cost of its purification and short catalytic half-life. This paper reports the development of a cost-effective method for the production and immobilization of OPH in a pilot application in an enzyme bioreactor column for detoxification of paraoxon and coumaphos in contaminated wastewaters. A fusion between OPH and a cellulose binding domain that binds selectively to cellulose was generated to allow one-step purification and immobilization of OPH on cheap and abundantly available cellulose immobilization matrices. When packed in a column bioreactor, the immobilized fusion enzyme was able to completely degrade coumaphos up to a concentration of 0.2 mM. However, stirring of OPH immobilized on cellulose materials resulted in complete OP degradation of 1.5 mM coumaphos. The bioreactor column degraded the compounds tested at high concentration, rapidly, and without loss of process productivity for about 2 months.

     

Reactive oxygen species regulate alkaloid metabolism in undifferentiated N. tabacum cells

Plants produce an immense number of natural products and undifferentiated cells from various plant tissues have long been considered an ideal source for their synthesis. However, undifferentiated plant cells often either lose their biosynthetic capacity over time or exhibit immediate repression of the required pathways once dedifferentiated. In this study, freshly prepared callus tissue was employed to further investigate the regulation of a natural product pathway in undifferentiated tobacco cells. Putrescine N-methyltransferase (PMT) is a pathway-specific enzyme required in nicotinic alkaloid production in Nicotiana species. Callus derived from transgenic Nicotiana tabacum plants harboring PMT promoter–GUS fusions were used to study factors that influence PMT expression. Under normal callus growth conditions in the presence of light and auxin, PMT promoter activity was strongly repressed. Conversely, dark conditions and the absence of auxin were found to upregulate PMT promoter activity, with light being dominant to the repressive effects of auxin. Since reactive oxygen species (ROS) are known by-products of photosynthesis and have been implicated in signaling, their involvement was investigated in transgenic callus by treatment with the ROS scavenger, dimethylthiourea, or catalase. Under highly repressive conditions for alkaloid synthesis, including normal culture conditions in the light, both ROS scavengers resulted in significant induction of PMT promoter activity. Moreover, treatment of callus with catalase resulted in the upregulation of PMT promoter activity and alkaloid accumulation in this tissue. These results suggest that ROS impact the regulation of the alkaloid pathway in undifferentiated cells and have implications for regulation of the pathway in other plant tissues.     

Rhodococcus lactonase with organophosphate hydrolase (OPH) activity and His6-tagged OPH with lactonase activity: evolutionary proximity of the enzymes and new possibilities in their application

Decontamination of soils with complex pollution using natural strains of microorganisms is a matter of great importance. Here we report that oil-oxidizing bacteria Rhodococcus erythropolis AC-1514D and Rhodococcus ruber AC-1513D can degrade various organophosphorous pesticides (OP). Cell-mediated degradation of five different OP is apparently associated with the presence of N-acylhomoserine lactonase, which is pronouncedly similar (46–50 %) to the well-known enzyme organophosphate hydrolase (OPH), a hydrolysis catalyst for a wide variety of organophosphorous compounds. Additionally, we demonstrated the high lactonase activity of hexahistidine-tagged organophosphate hydrolase (His₆-OPH) with respect to various N-acylhomoserine lactones, and we determined the catalytic constants of His₆-OPH towards these compounds. These experimental data and theoretical analysis confirmed the hypothesis about the evolutionary proximity of OPH and lactonases. Using Rhodococcus cells, we carried out effective simultaneous biodegradation of pesticide paraoxon (88 mg/kg) and oil hydrocarbon hexadecane (6.3 g/kg) in the soil. Furthermore, the discovered high lactonase activity of His₆-OPH offers new possibilities for developing an efficient strategy of combating resistant populations of Gram-negative bacterial cells.     

Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants

 The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus, most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic and escape shoots. The selection of transgenic shoots could be easily favored by eliminating the escapes and/or by performing shoot-tip grafting of the transgenic buds soon after their origin. GFP-expressing shoots have also been observed in citrus explants co-cultivated with Agrobacterium but cultured in a medium without the selective agent kanamycin. This opens the possibility to rescue the transgenic sectors and to regenerate transgenic plants without using selectable marker genes conferring antibiotic or herbicide resistance, which is currently a topic of much discussion for the commercialization of transgenic plants. Received: 28 October 1998 / Accepted: 28 November 1998     

Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the k_cat/K_m value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.     

Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.     

Generation of a mutagenized organophosphorus hydrolase for the biodegradation of the organophosphate pesticides malathion and demeton‐S

Aims: The bacterial organophosphorus hydrolase (OPH) enzyme hydrolyses and detoxifies a broad range of toxic organophosphate pesticides and warfare nerve agents by cleaving the various phosphorus‐ester bonds (P–O, P–F, P–CN, P–S); however, OPH hydrolyses these bonds with varying efficiencies. The aim of this study was to generate a variant OPH enzyme with improved hydrolytic efficiency against the poorly hydrolysed P–S class of organophosphates. Methods and Results: The gene encoding OPH was sequentially mutated at specific codons by saturation mutagenesis and screened for improved activity against the P–S substrates demeton‐S methyl and malathion. Escherichia coli lysates harbouring the variants displayed up to 177‐ and 1800‐fold improvement in specific activity against demeton‐S methyl and malathion, respectively, compared to the wild‐type lysates. The specificity constants of the purified variant proteins were improved up to 25‐fold for demeton‐S methyl and malathion compared to the wild‐type. Activity was associated with organophosphate detoxification as the hydrolysed substrate lost the ability to inhibit acetylcholinesterase. The improved hydrolytic efficiency against demeton‐S translated to the improved ability to hydrolyse the warfare agent VX. Conclusions: OPH variant enzymes were generated that displayed significantly improved ability to hydrolyse and detoxify organophosphates harbouring the P–S bond. Significance and Impact of the Study: The long‐term goal is to generate an environmentally‐friendly enzyme‐mediated bioremediation approach for the removal of toxic organophosphate compounds in the environment.     

A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues

We introduced the oat adc cDNA into rice under the control of the constitutive maize ubiquitin 1 promoter. We studied molecularly and biochemically sixteen independent transgenic plant lines. Significant increases in mRNA levels, ADC enzyme activity and polyamines were measured in transgenic callus. These increases were not maintained in vegetative tissue or seeds in regenerated plants, with the exception of one lineage. This particular lineage showed very significant increases in putrescine preferentially in seeds (up to 10 times compared to wild type and controls transformed with the hpt selectable marker alone). We have demonstrated that in cereals such as rice, over-expression of the oat adc cDNA results in increased accumulation of polyamines at different stages of development. We have also demonstrated that strong constitutive promoters, such as the maize ubiquitin 1 promoter, are sufficient to facilitate heritable high-level polyamine accumulation in seed. Our results demonstrate that by screening adequate numbers of independently derived transgenic plants, it is possible to identify those individuals which express a desired phenotype or genotype.     

Bacterial Cell Surface Display of Organophosphorus Hydrolase for Selective Screening of Improved Hydrolysis of Organophosphate Nerve Agents

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.     

药用植物栀子的组织培养

栀子(Gardenia jasminoides)为药用木本植物。以栀子果皮、种子团和种子为外植体,研究不同激素配比及不同培养方式对愈伤组织诱导和芽分化的影响。研究结果表明,培养基成分为MS+0.5 mg·L–12,4-D+0.25 mg·L–16-BA较适宜果皮和种子愈伤组织的诱导,诱导率分别为83.3%和88.5%;培养基成分为MS+1.0 mg·L–12,4-D+1.0 mg·L–16-BA较适宜种子团愈伤组织的诱导,诱导率为78.1%。3种外植体诱导的愈伤组织中,只有种子愈伤组织能通过液体培养分化出芽;TDZ对芽分化有明显的促进作用;最佳的芽分化培养基为MS+0.05 mg·L–1NAA+0.10 mg·L–1TDZ,其愈伤组织分化率为8.75%。该研究以栀子种子为外植体,并获得了再生植株,为药用植物栀子转基因体系的建立奠定了基础。     

A protocol for consistent, large-scale production of fertile transgenic rice plants

A protocol for consistent production of fertile transgenic rice plants was established utilizing microparticle bombardment of embryogenic tissues (Oryza sativa L. japonica cv. Taipei 309). This system has been employed to produce several thousand independently transformed plant lines carrying the hygromycin phosphotransferase (hph) gene and various genes of interest. The most efficient target tissue was highly embryogenic callus or suspension cell aggregates, when they were given an osmotic pre- and post-transformation treatment of 0.6 m carbohydrate. By optimizing the age of the tissue at the time of gene transfer and applying an improved selection procedure, transgenic plants were recovered in 8 weeks from the time of gene transfer, at an average of 22.3±9.7 per 100 calli and 22.4±8.0 plant lines per dish of suspension cell aggregates. This system has facilitated a number of studies using rice as a model for genetic transformation and will enable the large-scale production of transgenic rice plants for genomic studies. Received: 12 March 1998 / Revision received: 5 May 1998 / Accepted: 15 May 1998