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An engineered plant that accumulates higher levels of heavy metals than Thlaspi caerulescens, with yields of 100 times more biomass in mine soils.

Nicotiana glauca transformed with TaPCS1 was tested for its application in phytoremediation. When plantlets were grown in mine soils containing Cu, Zn, and Pb (42, 2600, and 1500 mg kg(-1)) the plant showed high levels of accumulation especially of Zn and Pb. Adult plants growing in mine soils containing different heavy metal concentrations showed a greater accumulation as well as an extension to a wider range of elements, including Cd, Ni and B. The overexpressed gene confers up to 9 and 36 times more Cd and Pb accumulation in the shoots under hydroponic conditions, and a 3- and 6-fold increase in mining soils. When the hyperaccumulator Thlaspi caerulescens was compared, the results were higher values of heavy metal and Boron accumulation, with a yield of 100 times more biomass. Thlaspi was unable to survive in mining soils containing either a level higher than 11000 mg kg(-1) of Pb and 4500 mg kg(-1) of Zn, while engineered plants yielded an average of 0.5 kg per plant.

Biodegradation, Environmental↗

Engineering plants with increased levels of the antioxidant chlorogenic acid.

The trend to view many foods not only as sustenance but also as medicine, so-called functional foods, is increasing. Phenolics are the most widespread dietary antioxidants, and among these, chlorogenic acid (CGA) accumulates to high levels in some crop plants. CGA acts as an antioxidant in plants and protects against degenerative, age-related diseases in animals when supplied in their diet. cDNA clones encoding the enzyme that synthesizes CGA, hydroxycinnamoyl-CoA quinate: hydroxycinnamoyl transferase (HQT), were characterized from tomato and tobacco. Gene silencing proved HQT to be the principal route for accumulation of CGA in solanaceous species. Overexpression of HQT in tomato caused plants to accumulate higher levels of CGA, with no side-effects on the levels of other soluble phenolics, and to show improved antioxidant capacity and resistance to infection by a bacterial pathogen. Tomatoes with elevated CGA levels could be used in foods with specific benefits for human health.

Acyl Coenzyme A↗

Field tests on managing resistance to Bt-engineered plants.

Several important crops have been engineered to express toxins of Bacillus thuringiensis (Bt) for insect control. In 1999, US farmers planted nearly 8 million hectares (nearly 20 million acres) of transgenic Bt crops approved by the EPA. Bt-transgenic plants can greatly reduce the use of broader spectrum insecticides, but insect resistance may hinder this technology. Present resistance management strategies rely on a "refuge" composed of non-Bt plants to conserve susceptible alleles. We have used Bt-transgenic broccoli plants and the diamondback moth as a model system to examine resistance management strategies. The higher number of larvae on refuge plants in our field tests indicate that a "separate refuge" will be more effective at conserving susceptible larvae than a "mixed refuge" and would thereby reduce the number of homozygous resistant (RR) offspring. Our field tests also examined the strategy of spraying the refuge to prevent economic loss to the crop while maintaining susceptible alleles in the population. Results indicate that great care must be taken to ensure that refuges, particularly those sprayed with efficacious insecticides, produce adequate numbers of susceptible alleles. Each insect/Bt crop system may have unique management requirements because of the biology of the insect, but our studies validate the need for a refuge. As we learn more about how to refine our present resistance management strategies, it is important to also develop the next generation of technology and implementation strategies.

Animals↗

Re-engineering plant gene targeting.

The genome sequence of Arabidopsis is complete and the genomes of plants representing legumes (Medicago truncatula) and grasses (rice) will soon follow. The rate at which new genes have been discovered has far outstripped the pace at which their function is determined. The greatest hurdle that plant biologists face in assigning gene function and in crop improvement is the lack of efficient and robust technologies to generate gene replacements or targeted gene knockouts. Many of the factors underlying these events remain to be elucidated. This review addresses the current status of plant gene targeting and what is known about the associated plant DNA repair mechanisms.

DNA, Plant↗

The similarities of bar and pat gene products make them equally applicable for plant engineers.

The bar and pat genes, isolated from different Streptomyces species, both encode a phosphinothricin acetyltransferase (PAT) and are widely applied in plant genetic engineering. The genes were expressed in Escherichia coli and the corresponding proteins were purified and used for functional and structural comparison. Both proteins are homodimers regardless of whether they are expressed in microorganisms or in plants. They have comparable molecular weights and show immuno-cross-reactivity to their respective polyclonal antisera. The enzymes have a similar substrate affinity towards L-phosphinothricin and do not acetylate any of the other L-amino acids tested. In model digestion experiments using simulated human gastric fluids, their enzymatic activity is decreased within seconds, accompanied by a rapid and complete breakdown of both proteins. These data demonstrate the structural and functional equivalence of the PAT proteins, which is also reflected in the comparable performance of transgenic plants carrying the bar or pat gene.

Acetyltransferases↗

[Plant lectin and its application in insect-resistant plant genetic engineering].

Plant lectins are proteins possessing at least one non-catalytic domain that binds reversibly to specific mono-or oligosaccharides. They distinguish themselves from other plant proteins by the ability of carbohydrate binding. Most plant lectins are directed to bind foreign polysaccharide. Plant lectin is believed to take part in the defense responses against invader. In this paper we presented a review on the classification, characters, functions, crystal structure and,functions related to the carbohydrate binding activity. The status and prospect of plant lectins utilization were also discussed.

English Abstract↗

Integrating genomics and metabolomics for engineering plant metabolic pathways.

Plant metabolites are characterized by an enormous chemical diversity, every plant having its own complex set of metabolites. This variety poses analytical challenges, both for profiling multiple metabolites in parallel and for the quantitative analysis of selected metabolites. We are only just starting to understand the roles of these metabolites, many of them being involved in adaptations to specific ecological niches and some finding beneficial use (e.g. as pharmaceuticals). Spectacular advances in plant metabolomics offer new possibilities, together with the aid of systems biology, to explore the extraordinary complexity of the plant biochemical capacity. State-of-the art genomics tools can be combined with metabolic profiling to identify key genes that could be engineered for the production of improved crop plants.

Arabidopsis↗

Through the lens of bioenergy crops: advances, bottlenecks, and promises of plant engineering.

Advances in engineering of bioenergy crops were driven over the past years by adapting technological breakthroughs and accelerating conventional applications but also exposed intriguing challenges. New tools revealed rich interconnectivity in the exponentially growing and dynamic 'big' omics data' of metabolomes, transcriptomes, and genomes at previously inaccessible magnitude (global, cross-species, meta-) and resolution (single cell). Insights enabled fresh hypotheses and stimulated disciplines such as functional genomics with discovery of broad regulatory networks and their determinants, that is, DNA parts, including promoters, regulatory elements, and transcription factors. Their rational design, assembly into increasingly complex blueprints, and installation into diverse chassis is an existing frontier that may benefit from emerging technologies to address bottlenecks. Interweaving nature-inspired to fully synthetic parts has already allowed building of fine-tuned regulatory circuits, or new-to-nature metabolic routes insulated from the biological context of the chassis species. Similarly, developments and the evolving need for unifying principles in plant transformation and species-agnostic technologies highlight future opportunities for engineering the next generation of bioenergy plants.

Crops, Agricultural↗

Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection.

Chloroplast genetic engineering offers several advantages over nuclear transformation including high levels of gene expression and gene containment. However, a consequence of placing a transgene in the chloroplast genome is that the antibiotic resistance genes used as selectable markers are highly amplified. Engineering genetically modified (GM) crops without the use of antibiotic resistance genes should eliminate the potential risk of their transfer to the environment or gut microbes. Therefore, the betaine aldehyde dehydrogenase (BADH) gene from spinach was used in this study as a selectable marker. The selection process involves conversion of toxic betaine aldehyde (BA) by the chloroplast BADH enzyme to non-toxic glycine betaine, which also serves as an osmoprotectant. Chloroplast transformation efficiency was 25-fold higher in BA selection than with spectinomycin. In addition, rapid regeneration was obtained. Transgenic shoots appeared within 12 days in 80% of leaf disks (up to 23 shoots per disk) under BA selection compared to 45 days in 15% of disks (1 or 2 shoots per disk) under spectinomycin selection. Southern blots confirmed stable integration of foreign genes into all of the chloroplast genomes (approximately 10,000 copies per cell) resulting in homoplasmy. Transgenic tobacco plants showed 15- to 18-fold higher BADH activity at different developmental stages than untransformed controls. Transgenic plants were morphologically indistinguishable from untransformed plants and the introduced trait was inherited stably in the subsequent generation. This is the first report of genetic engineering of the higher plant chloroplast genome without the use of antibiotic selection. The use of naturally occurring genes in spinach for selection, in addition to gene containment, should ease public concerns regarding GM crops.

Alcohol Oxidoreductases↗

Engineering plants for elevated CO(2): a relationship between starch degradation and sugar sensing.

In the future, plants will have additional CO(2) for photosynthesis. However, plants do not take maximal advantage of this additional CO(2) and it has been hypothesized that end product synthesis limitations and sugar sensing mechanisms are important in regulating plant responses to increasing CO(2). Attempts to increase end product synthesis capacity by engineering increased sucrose-phosphate synthase activity have been generally, but not universally, successful. It was found that plants benefited from a two- to three-fold increase in SPS activity but a 10-fold increase did not increase yield. Despite the success in increasing yield, increasing SPS did not increase photosynthesis. However, carbon export from chloroplasts was increased during the day and reduced at night (when starch provides carbon for sucrose synthesis. We develop here a hypothesis that starch degradation is closely sensed by hexokinase because a newly discovered pathway required for starch to sucrose conversion that involves maltose is one of few metabolic pathways that requires hexokinase activity.

Biological Transport↗

Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations.

Abiotic stresses, especially salinity and drought, are the primary causes of crop loss worldwide. Plant adaptation to environmental stresses is dependent upon the activation of cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Consequently, engineering genes that protect and maintain the function and structure of cellular components can enhance tolerance to stress. Our limited knowledge of stress-associated metabolism remains a major gap in our understanding; therefore, comprehensive profiling of stress-associated metabolites is most relevant to the successful molecular breeding of stress-tolerant crop plants. Unraveling additional stress-associated gene resources, from both crop plants and highly salt- and drought-tolerant model plants, will enable future molecular dissection of salt-tolerance mechanisms in important crop plants.

Adaptation, Physiological↗

A long way ahead: understanding and engineering plant metal accumulation.

Some plants can hyperaccumulate metal ions that are toxic to virtually all other organisms at low dosages. This trait could be used to clean up metal-contaminated soils. Moreover, the accumulation of heavy metals by plants determines both the micronutrient content and the toxic metal content of our food. Complex interactions of transport and chelating activities control the rates of metal uptake and storage. In recent years, several key steps have been identified at the molecular level, enabling us to initiate transgenic approaches to engineer the transition metal content of plants.

Biodegradation, Environmental↗