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PubMed2003

Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content.

Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux.

Alkyl and Aryl Transferases↗
PubMed2003

Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity.

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.

Arabidopsis↗
PubMed2000

Engineering direct fructose production in processed potato tubers by expressing a bifunctional alpha-amylase/glucose isomerase gene complex.

Manipulation of starch biosynthesis/degradation and formation of novel molecules in storage organs of plants through genetic engineering is an attractive but technically challenging goal. We report here, for the first time, that starch was degraded and glucose and fructose were produced directly when crushed potato tubers expressing a starch degrading bifunctional gene were heated for 45 minutes at 65 degrees C. To achieve this, we have constructed a fusion gene encoding the thermostable enzymes: alpha-amylase (Bacillus stearothermophilus) and glucose isomerase (Thermus thermophilus). The chimeric gene was placed under the control of the granule-bound-starch synthase promoter. This enzymatic complex produced in transgenic tubers was only active at high temperature (65 degrees C). More than 100 independent transgenic potato plants were regenerated. Molecular analyses confirmed the stable integration of the chimeric gene into the potato genome. The biochemical analyses performed on young and old tubers after high-temperature treatment (65 degrees C) revealed an increase in the formation rate of fructose and glucose by a factor of 16.4 and 5. 7, respectively, in the transgenic tubers as compared to untransformed control tubers. No adverse discernible effect on plant development and metabolism including tuber formation and starch accumulation was observed in the transgenic plants before heat treatment. Our results demonstrate that it is possible to replace starch degradation using microbial enzymes via a system where the enzymes are produced directly in the plants, but active only at high temperature, thus offering novel and viable strategies for starch-processing industries.

Aldose-Ketose Isomerases↗
PubMed1992

Evaluation of methods for detecting ecological effects from genetically engineered microorganisms and microbial pest control agents in terrestrial systems.

This report summarizes and evaluates research from several laboratories that deals with the detection of ecological effects induced through exposure of microbes or plants to genetically engineered microorganisms (GEMs) and microbial pest control agents (MPCAs). Some 27 potential endpoints for measuring effects have been studied. Perturbations induced by GEMs have been detected in about one-half of these endpoints. Detectable effects have been recorded for over half of the 16 species of bacteria and fungi studied. The effects caused by GEMs and MPCAs include inhibition of beneficial mycorrhizal fungi growing on Douglas fir seedling roots, depression in plant root and shoot growth, inhibition of predatory soil protozoa, accumulation of a toxic metabolite during biodegradation that inhibits soil fungi, increased microbial community respiration due to rapid lignin breakdown in soil, and the displacement of a broad group of gram-negative bacteria that inhabit the root surface of cereal crops. These effects were usually, but not always, of short duration. However, some of the changes were irreversible during the observation time of days, weeks, or in one case, months.

Journal Article↗
PubMed1999

T-DNA transfer to maize plants.

Agrobacterium-mediated transformation is the method of choice to engineer desirable genes into plants. Here we describe a protocol for demonstrating T-DNA transfer from Agrobacterium into the economically important graminaceous plant maize. Expression of the T-DNA-located GUS gene was observed with high efficiency on shoots of young maize seedlings after cocultivation with Agrobacterium.

DNA, Bacterial↗
PubMed2000

Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides.

Maize plants resistant to imidazolinone herbicides were engineered through targeted modification of endogenous genes using chimeric RNA/DNA oligonucleotides. A precise single-point mutation was introduced into genes encoding acetohydroxyacid synthase (AHAS), at a position known to confer imidazolinone resistance. Phenotypically normal plants from the converted events (C0) were regenerated from resistant calli and grown to maturity. Herbicide leaf painting confirmed the resistance phenotype in C0 plants and demonstrated the anticipated segregation pattern in C1 progeny. DNA cloning and sequencing of the targeted region in resistant calli and derived C0 and C1 plants confirmed the expected mutation. These results demonstrate that oligonucleotide-mediated gene manipulation can be applied to crop improvement. This approach does not involve genomic integration of transgenes. Since the new trait is obtained through modifying a gene within its normal chromosomal context, position effects, transgene silencing, or other concerns that arise as part of developing transgenic events are avoided.

Acetolactate Synthase↗
PubMed2005

Selenium uptake, translocation, assimilation and metabolic fate in plants.

The chemical and physical resemblance between selenium (Se) and sulfur (S) establishes that both these elements share common metabolic pathways in plants. The presence of isologous Se and S compounds indicates that these elements compete in biochemical processes that affect uptake, translocation and assimilation throughout plant development. Yet, minor but crucial differences in reactivity and other metabolic interactions infer that some biochemical processes involving Se may be excluded from those relating to S. This review examines the current understanding of physiological and biochemical relationships between S and Se metabolism by highlighting their similarities and differences in relation to uptake, transport and assimilation pathways as observed in Se hyperaccumulator and non-accumulator plant species. The exploitation of genetic resources used in bioengineering strategies of plants is illuminating the function of sulfate transporters and key enzymes of the S assimilatory pathway in relation to Se accumulation and final metabolic fate. These strategies are providing the basic framework by which to resolve questions relating to the essentiality of Se in plants and the mechanisms utilized by Se hyperaccumulators to circumvent toxicity. In addition, such approaches may assist in the future application of genetically engineered Se accumulating plants for environmental renewal and human health objectives.

Animals↗
PubMed2000

The generation of plant disease resistance gene specificities.

We are gaining an understanding of the molecular basis of resistance specificity and of the natural processes that generate different specificities. This is a prerequisite for the genetic engineering of new plant disease-resistance genes to control diseases for which naturally occurring resistance is inadequate. DNA sequence analysis indicates that point mutation, recombination and selection can generate and maintain the high levels of polymorphism observed in resistance genes. Comparisons of closely related resistance proteins indicate that specificity can be determined by variation in at least two regions. One of these contains leucine-rich repeats, which are a common feature of most resistance proteins.

Amino Acid Sequence↗
PubMedDate not supplied

"This food may contain ..." What nurses should know about genetically engineered foods.

Genetic engineering has been in existence since 1973. The process involves placing genetic DNA from one organism into another. Genetically engineered organisms (GEOs) are the name given to such new species of plants created through this process. Proponents of GEOs assert that foods we are now able to produce have greater nutritional value, longer shelf life, better appearance, taste and smell. There are positive benefits to genetic engineering of plants and animals. A growing concern for the health safety of genetically engineered plants and foods is developing among the cautious. The purpose of this article is to define genetic engineering, present benefits and risks, describe the impact on human health, and address implications for nursing.

Consumer Product Safety↗
PubMed2003

Plant molecular biology and biotechnology research in the post-recombinant DNA era.

After the beginning of the recombinant DNA era in the mid-1970s, researchers in India started to make use of the new technology to understand the structure of plant genes and regulation of their expression. The outcome started to appear in print in early the 1980s and genes for histones, tubulin, photosynthetic membrane proteins, phototransduction components, organelles and those regulated differentially by developmental and extrinsic signals were sequenced and characterized. Some genes of biotechnological importance like those encoding an interesting seed protein and the enzyme glyoxalase were also isolated. While work on the characterization of genome structure and organization was started quite early, it remained largely focused on the identification of DNA markers and genetic variability. In this context, the work on mustard, rice and wheat is worth mentioning. In the year 2000, India became a member of the international consortium to sequence entire rice genome. Several laboratories have also given attention to regulated expression of plastid and nuclear genes as well as to isolate target-specific promoters or design promoters with improved potential. Simultaneously, transgenic systems for crops like mustard, rice, wheat, cotton, legumes and several vegetables have been established. More recently, genes of agronomic importance like those for insect resistance, abiotic stress tolerance, nutritional improvement and male sterility, isolated in India or abroad, have been utilized for raising transgenics for crop improvement. Some of these transgenics have already shown their potential in containment facility or limited field trials conducted under the stipulated guidelines. Plant molecular biology and biotechnology are thus clearly poised to make an impact on research in basic biology and agriculture in the near future.

Agriculture↗
PubMed1994

Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability.

Most starch hydrolases and related enzymes belong to the alpha-amylase family which contains a characteristic catalytic (beta/alpha)8-barrel domain. Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme. Crystal structures have been reported in three of these enzyme classes: the alpha-amylases, the cyclodextrin glucanotransferases, and the oligo-1,6-glucosidases. Throughout the alpha-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the beta-->alpha loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of alpha-amylases, changed the distribution of alpha-, beta- and gamma-cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative alpha-1,4:alpha-1,6 dual-bond specificity of neopullulanase. Barley alpha-amylase isozyme hybrids and Bacillus alpha-amylases demonstrate the impact of a small domain B protruding from the (beta/alpha)8-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as alpha-amylase, starch branching and debranching enzymes, and amylomaltase.

Amino Acid Sequence↗
PubMed2004

Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration.

Hairy root cultures of Catharanthus roseus var. Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents. On the basis of growth pattern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories. However based on the integration of the Ri T(L)-DNA and T(R)-DNA genes, there were only three different categories of independent hairy root clones-C1 (rolA&B(+)/ags(+)), C2 (rolA&B(-)/ags(+)) and C3 (rolA&B(+)/ags(-)). Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones. The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the T(L)-DNA and T(R)-DNA genes (C1) and the T(L)-DNA gene (C3) alone. Two rolA&B(-) but ags(+) clones (C2) accumulated much less or only very negligible amounts of ajmalicine. The possible role of the T(L)-DNA and T(R)-DNA genes on growth and alkaloid accumulation in these root clones is discussed.

Alkaloids↗