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PubMed2005

Phytoremediation of toxic trace elements in soil and water.

Toxic heavy metals and metalloids, such as cadmium, lead, mercury, arsenic, and selenium, are constantly released into the environment. There is an urgent need to develop low-cost, effective, and sustainable methods for their removal or detoxification. Plant-based approaches, such as phytoremediation, are relatively inexpensive since they are performed in situ and are solar-driven. In this review, we discuss specific advances in plant-based approaches for the remediation of contaminated water and soil. Dilute concentrations of trace element contaminants can be removed from large volumes of wastewater by constructed wetlands. We discuss the potential of constructed wetlands for use in remediating agricultural drainage water and industrial effluent, as well as concerns over their potential ecotoxicity. In upland ecosystems, plants may be used to accumulate metals/metalloids in their harvestable biomass (phytoextraction). Plants can also convert and release certain metals/metalloids in a volatile form (phytovolatilization). We discuss how genetic engineering has been used to develop plants with enhanced efficiencies for phytoextraction and phytovolatilization. For example, metal-hyperaccumulating plants and microbes with unique abilities to tolerate, accumulate, and detoxify metals and metalloids represent an important reservoir of unique genes that could be transferred to fast-growing plant species for enhanced phytoremediation. There is also a need to develop new strategies to improve the acceptability of using genetically engineered plants for phytoremediation.

Biodegradation, Environmental↗
PubMed1996

Engineered GFP as a vital reporter in plants.

BACKGROUND: The green-fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently been used as a universal reporter in a broad range of heterologous living cells and organisms. Although successful in some plant transient expression assays based on strong promoters or high copy number viral vectors, further improvement of expression efficiency and fluorescent intensity are required for GFP to be useful as a marker in intact plants. Here, we report that an extensively modified GFP is a versatile and sensitive reporter in a variety of living plant cells and in transgenic plants. RESULTS: We show that a re-engineered GFP gene sequence, with the favored codons of highly expressed human proteins, gives 20-fold higher GFP expression in maize leaf cells than the original jellyfish GFP sequence. When combined with a mutation in the chromophore, the replacement of the serine at position 65 with a threonine, the new GFP sequence gives more than 100-fold brighter fluorescent signals upon excitation with 490 nm (blue) light, and swifter chromophore formation. We also show that this modified GFP has a broad use in various transient expression systems, and allows the easy detection of weak promoter activity, visualization of protein targeting into the nucleus and various plastids, and analysis of signal transduction pathways in living single cells and in transgenic plants. CONCLUSIONS: The modified GFP is a simple and economical new tool for the direct visualization of promoter activities with a broad range of strength and cell specificity. It can be used to measure dynamic responses of signal transduction pathways, transfection efficiency, and subcellular localization of chimeric proteins, and should be suitable for many other applications in genetically modified living cells and tissues of higher plants. The data also suggest that the codon usage effect might be universal, allowing the design of recombinant proteins with high expression efficiency in evolutionarily distant species such as humans and maize.

Allium↗
PubMed1999

Flavonoids and isoflavonoids - a gold mine for metabolic engineering.

Flavonoid-derived plant natural products have long been known to function as floral pigments for the attraction of insect pollinators, as signal molecules for beneficial microorganisms in the rhizosphere, and as antimicrobial defense compounds. New functions for flavonoid compounds continue to be found, particularly in plant-microorganism signaling, and there has been an explosion of interest in flavonoids and isoflavonoids as health-promoting components of the human diet. The flavonoid and isoflavonoid pathways are probably the best characterized natural product pathway in plants, and are therefore excellent targets for metabolic engineering. Manipulation of flavonoid biosynthesis can be approached via several strategies, including sense or antisense manipulation of pathway genes, modification of the expression of regulatory genes, or generation of novel enzymatic specificities by ra-tional approaches based on emerging protein structure data. In addition, activation tagging provides a novel approach for the discovery of uncharacterized structural and regulatory genes of flavonoid biosynthesis.

Journal Article↗
PubMed2004

Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis.

Plants are the main source of folate in human diets, but many fruits, tubers, and seeds are poor in this vitamin, and folate deficiency is a worldwide problem. Plants synthesize folate from pteridine, p-aminobenzoate (PABA), and glutamate moieties. Pteridine synthesis capacity is known to drop in ripening tomato fruit; therefore, we countered this decline by fruit-specific overexpression of GTP cyclohydrolase I, the first enzyme of pteridine synthesis. We used a synthetic gene based on mammalian GTP cyclohydrolase I, because this enzyme is predicted to escape feedback control in planta. This engineering maneuver raised fruit pteridine content by 3- to 140-fold and fruit folate content by an average of 2-fold among 12 independent transformants, relative to vector-alone controls. Most of the folate increase was contributed by 5-methyltetrahydrofolate polyglutamates and 5,10-methenyltetrahydrofolate polyglutamates, which were also major forms of folate in control fruit. The accumulated pteridines included neopterin, monapterin, and hydroxymethylpterin; their reduced forms, which are folate biosynthesis intermediates; and pteridine glycosides not previously found in plants. Engineered fruit with intermediate levels of pteridine overproduction attained the highest folate levels. PABA pools were severely depleted in engineered fruit that were high in folate, and supplying such fruit with PABA by means of the fruit stalk increased their folate content by up to 10-fold. These results demonstrate that engineering a moderate increase in pteridine production can significantly enhance the folate content in food plants and that boosting the PABA supply can produce further gains.

Aldehyde-Lyases↗
PubMed1990

Intra- and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase.

A site-specific recombination system has many potential uses for rearranging genetic material in higher eukaryotic cells: for example, the control of gene expression by deletion or inversion of DNA segments, the clustering of transgenic constructs via site-specific integration, and the generation of chromosomal translocations. In this report, we describe a first step towards the application of a site-specific recombination system in plant cells. By use of a transient assay, we demonstrate that the bacteriophage P1 cre gene can be expressed as a functional recombinase in tobacco cells. Upon expression in tobacco protoplasts, Cre recognizes its target sites, lox, and mediates reciprocal genetic crossovers at these sites. When the lox sites are present in cis to one another, and arranged in either direct or inverted orientations, we detect Cre/lox-specific deletion and inversion events, respectively. The placement of lox sites in trans resulted in the co-integration of the substrates by Cre-mediated intermolecular recombination. These results indicate that the Cre/lox site-specific recombination system might be further developed as an additional tool for manipulating DNA in plant cells. Applications relevant to the genetic engineering of higher plants are discussed.

Base Sequence↗
PubMed2005

New vectors and marker excision systems mark progress in engineering the plastid genome of higher plants.

The transformation of the plastid genome, until recently restricted to tobacco, is now being extended to a rapidly growing list of crops. This perspective provides an overview of emerging trends of technology development in the field with a focus on vector design and marker excision systems. The new tools will facilitate engineering of the photosynthetic machinery and enable novel agricultural and industrial applications.

Genes, Reporter↗
PubMed2006

Engineering ketocarotenoid biosynthesis in potato tubers.

Consumption of astaxanthin is increasingly associated with a range of health benefits. Attempts to engineer ketocarotenoid biosynthesis in plants have been successful although there are no reports of nutritionally significant levels of astaxanthin in plant storage organs. Thus, in this study, ketocarotenoid biosynthesis was engineered in potato tubers. Both Solanum tuberosum and Solanum phureja transgenic lines were produced that expressed an algal bkt1 gene, encoding a beta-ketolase, and accumulated ketocarotenoids. Two major ketocarotenoids were detected, ketolutein and astaxanthin. The level of unesterified astaxanthin reached ca. 14 microg g(-1) DW in some bkt1 expressing lines of S. phureja but was much lower in the S. tuberosum background. Co-transformation of S. tuberosum with crtB, encoding phytoene synthase, and the bkt1 gene was achieved in order to determine whether this would enhance the levels of S. tuberosum ketocarotenoid.

Carotenoids↗
PubMed1993

Genetic engineering of commercially useful biosynthetic pathways in transgenic plants.

In many economically important plant species, the chemical composition of one or more non-protein compounds determines the value of the plant and may have an important role in protecting the plant from environmental stress, including pests, drought, salt, temperature and light. A number of potential opportunities exist whereby the range or amount of such valuable compounds can be increased by genetic engineering.

Carbohydrates↗
PubMed1996

Engineering strategies for the design of plant nutrient delivery systems for use in space: approaches to countering microbiological contamination.

Microbiological contamination of crops within space-based plant growth research chambers has been postulated as a potentially significant problem. Microbial infestations; fouling of Nutrient Delivery System (NDS) fluid loops; and the formation of biofilms have been suggested as the most obvious and important manifestations of the problem. Strict sanitation and quarantine procedures will reduce, but not eliminate, microbial species introduced into plant growth systems in space habitats. Microorganisms transported into space most likely will occur as surface contaminants on spacecraft components, equipment, the crew, and plant-propagative materials. Illustrations of the potential magnitude of the microbiological contamination issue will be drawn from the literature and from documentation of laboratory and commercial field experience. Engineering strategies for limiting contamination and for the development of countermeasures will be described. Microbiological control technologies and NDS hardware will be discussed. Configurations appropriate for microgravity research facilities, as well as anticipated bio-regenerative life support system implementations, will be explored. An efficiently designed NDS, capable of adequately meeting the environmental needs of crop plants in space, is considered to be critical in both the research and operational domains. Recommended experiments, tests, and technology developments, structured to allow the development of prudent engineering solutions also will be presented.

Biofilms↗
PubMed2003

Plant cytochromes P450: tools for pharmacology, plant protection and phytoremediation.

Cytochromes P450 catalyse extremely diverse and often complex regiospecific and/or stereospecific reactions in the biosynthesis or catabolism of plant bioactive molecules. Engineered P450 expression is needed for low-cost production of antineoplastic drugs such as taxol or indole alkaloids and offers the possibility to increase the content of nutraceuticals such as phytoestrogens and antioxidants in plants. Natural products may serve important functions in plant defence and metabolic engineering of P450s is a prime target to improve plant defence against insects and pathogens. Herbicides, pollutants and other xenobiotics are metabolised by some plant P450 enzymes. These P450s are tools to modify herbicide tolerance, as selectable markers and for bioremediation.

Antineoplastic Agents↗
PubMed1999

Phytoremediation of methylmercury pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials.

Methylmercury is an environmental toxicant that biomagnifies and causes severe neurological degeneration in animals. It is produced by bacteria in soils and sediments that have been contaminated with mercury. To explore the potential of plants to extract and detoxify this chemical, we engineered a model plant, Arabidopsis thaliana, to express a modified bacterial gene, merBpe, encoding organomercurial lyase (MerB) under control of a plant promoter. MerB catalyzes the protonolysis of the carbon---mercury bond, removing the organic ligand and releasing Hg(II), a less mobile mercury species. Transgenic plants expressing merBpe grew vigorously on a wide range of concentrations of monomethylmercuric chloride and phenylmercuric acetate. Plants lacking the merBpe gene were severely inhibited or died at the same organomercurial concentrations. Six independently isolated transgenic lines produced merBpe mRNA and MerB protein at levels that varied over a 10- to 15-fold range, and even the lowest levels of merBpe expression conferred resistance to organomercurials. Our work suggests that native macrophytes (e.g., trees, shrubs, grasses) engineered to express merBpe may be used to degrade methylmercury at polluted sites and sequester Hg(II) for later removal.

Air Pollutants↗
PubMedDate not supplied

Engineering control technology in polyvinyl chloride polymerization plants.

The National Institute for Occupational Safety and Health, Division of Physical Sciences and Engineering has initiated a research program in control technology. The objective of this program is to facilitate the implementation of effective preventative measures in order to prevent occupational illness. The plastics and resins industry control technology assessment has recently been completed. The objectives of this study were to document and evaluate effective control technology for plastics and resins polymerization plants. Particular emphasis was given to PVC polymerization processes, since the relatively recent lowering in the personal exposure limit for vinyl chloride monomer (VCM) to an 8-hour 1-ppm time-weighted average has required the application of state-of-the-art controls. The present paper contains a summary of the control technology that was found to be effective in controlling VCM in processes manufacturing PVC by suspension, bulk, and dispersion polymerization. Controls necessary for VCM include process and equipment modification, isolation, local and general ventilation, work practices, personal protective equipment, workplace monitoring systems, employee/employer education, and on-going effort by both workers and management. All of these components must function together as an integrated coordinated system in order to assure worker protection under normal operating conditions or under conditions of process upset or maintenance.

Chemical Industry↗
PubMed1991

[Assessment of the functional status of the engineering-technical staff at a machine-building plant].

For early detection of functional disorders and risk-factors among the engineering staff at a machine-building plant, a set of highly informative, physiologically grounded and accessible techniques were used. To improve the patterns of out-patient screening of the staff, the contributors used physiologic and psychologic testing techniques, ECG monitoring registration in dynamics in the real occupational conditions, and riskometry, which facilitated the substantiation procedures for individual medical recommendations.

Adult↗
PubMed1993

Isolating plant genes.

The genetic transformation of most agriculturally important plant species is now possible. However, the application of this technology to rational plant-improvement is currently limited by a shortage of cloned genes for important traits. Recent technological advances in plant-gene isolation and identification, such as map-based cloning, insertional mutagenesis and large-scale cDNA sequencing, have accelerated the rate of gene isolation and significantly expanded the opportunities for genetic engineering of crop plants.

Biotechnology↗
PubMed2000

Novel Approaches to Oral Vaccines: Delivery of Antigens by Edible Plants.

Advances in genetic engineering in the past decade have accelerated the expression, in plants, of foreign proteins with industrial and pharmaceutical value. Antigens from infectious bacterial or viral diseases have been introduced into plants through plant virus-mediated infection or Agrobacterium tumefaciens-mediated stable transformation methods. Oral immunization with transgenic plant tissues that contain vaccine antigen proteins stimulates both systemic and mucosal immune responses in animals. Plant-based vaccines can provide significant levels of protection against challenge by viral or bacterial pathogens.

Journal Article↗
PubMed2004

Metabolic engineering of ketocarotenoid formation in higher plants.

Although higher plants synthesize carotenoids, they do not possess the ability to form ketocarotenoids. In order to generate higher plants capable of synthesizing combinations of ketolated and hydroxylated carotenoids the genes responsible for the carotene 4,4' oxygenase and 3,3' hydroxylase have been transformed into tomato and tobacco. The gene products were produced as a polyprotein. Subsequent cleavage of the polyprotein, targeting of the two enzymes to the plastid and enzyme activities have been shown for both gene products. Metabolite profiling has shown the formation of ketolated carotenoids from beta-carotene and its hydroxylated intermediates in tobacco and tomato leaf. In the nectary tissues of tobacco flowers a quantitative increase (10-fold) as well as compositional changes were evident, including the presence of astaxanthin, canthaxanthin and 4-ketozeaxanthin. Interestingly, in this tissue the newly formed carotenoids resided predominantly as esters. These data are discussed in terms of metabolic engineering of carotenoids and their sequestration in higher plant tissues.

Bacterial Proteins↗