The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use. Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD 50 value approximately 2-to 4-fold greater than the sensitive biotype collected from the same region. A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC 50 (glyphosate) for the (R) biotype. Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes. One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium. Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity. Escherichia coli aroA (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype. These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species.

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). ² These authors contributed equally to this work. SummaryPlastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodi®ed prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the ef®cacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the ef®cacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identi®ed translational control sequences that direct a 10 000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for ef®cacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

Poly(hydroxyalkanoates) are natural polymers with thermoplastic properties. One polymer of this class with commercial applicability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) can be produced by bacterial fermentation, but the process is not economically competitive with polymer production from petrochemicals. Poly(hydroxyalkanoate) production in green plants promises much lower costs, but producing copolymer with the appropriate monomer composition is problematic. In this study, we have engineered Arabidopsis and Brassica to produce PHBV in leaves and seeds, respectively, by redirecting the metabolic flow of intermediates from fatty acid and amino acid biosynthesis. We present a pathway for the biosynthesis of PHBV in plant plastids, and also report copolymer production, metabolic intermediate analyses, and pathway dynamics.

Glyphosate is a broad-spectrum herbicide that has been used extensively for more than 20 yr. The first glyphosate-resistant weed biotype appeared in 1996; it involved a rigid ryegrass population from Australia that exhibited an LD50 value approximately 10-fold higher than that of sensitive biotypes. We have characterized gene expression levels and glyphosate sensitivity of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), the target enzyme for glyphosate inhibition, in sensitive and resistant lines derived from this population. Restriction fragment length polymorphism analyses were also performed to examine the distribution of EPSPS gene variants and the gene copy number. A two- to threefold increase in basal EPSPS messenger RNA (mRNA) and enzyme activity levels was observed in the most resistant lines analyzed; however, differences among lines in the sensitivity of EPSPS to glyphosate were not apparent. Induction of EPSPS was observed within 48 h after application of 1.5 kg ae ha−1 of glyphosate. This was reflected in elevated levels of both EPSPS mRNA and enzyme activity. Similarly, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase mRNA levels increased after glyphosate treatment; however, basal and induced transcript levels were comparable for sensitive and resistant lines in this case. The restriction fragment length polymorphism analyses showed no evidence for gene amplification or cosegregation of a specific EPSPS gene variant with glyphosate resistance. EPSPS expression in lines exhibiting an intermediate level of resistance was indistinguishable from that in glyphosate-sensitive lines, suggesting that the mechanism could, at least in part, be non–target-based.

Many essential oils (EOs) have anticonvulsant activity and might benefit people with epilepsy. Lemongrass, lavender, clove, dill, and other EOs containing constituents such as asarone, carvone, citral, eugenol, or linalool are good candidates for evaluation as antiepileptic drugs. On the other hand, some EOs have convulsant effects and may trigger seizures in both epileptic and healthy individuals. Internal use of EOs like sage, hyssop, rosemary, camphor, pennyroyal, eucalyptus, cedar, thuja, and fennel can cause epileptic seizures because they contain thujone, 1,8-cineole, camphor, or pinocamphone, which have been identified as convulsive agents. While more research is needed to confirm their mechanisms of action, it appears that the convulsant or anticonvulsant properties of essential oils are largely due to (1) their ability to modulate the GABAergic system of neurotransmission and (2) their capacity to alter ionic currents through ion channels. This review presents a systematic analysis of the current research on EOs and epilepsy, including human case studies, animal models, andin vitrostudies.

The lack of alternative selectable markers in crop transformation has been a substantial barrier for commercial application of agricultural biotechnology. We have developed an efficient selection system for wheat transformation using glyphosate-tolerant CP4 and GOX genes as a selectable marker. Immature embryos of the wheat cultivar Bobwhite were bombarded with two separate plasmids harboring the CP4/GOX and GUS genes. After a 1 week delay, the bombarded embryos were transferred to a selection medium containing 2 mM glyphosate. Embryo-derived calli were subcultured onto the same selection medium every 3 weeks consecutively for 9-12 weeks, and were then regenerated and rooted on selection media with lower glyphosate concentrations. Transgenic plants tolerant to glyphosate were recovered. ELISA assay confirmed expression of the CP4 and GOX genes in R0 plants. Southern blot analysis demonstrated that the transgenes were integrated into the wheat genomes and transmitted to the following generation. The use of CP4 and GOX genes as a selectable marker provides an efficient, effective, and alternative transformation selection system for wheat.

Although frankincense essential oil (FREO) has become increasingly popular in skin care, research on its biological activities in human skin cells is scarce, if not completely absent. In the current study, we explored the biological activities of FREO in pre-inflamed human dermal fibroblasts by analyzing the levels of 17 important protein biomarkers pertinent to inflammation and tissue remodeling. FREO exhibited robust anti-proliferative activity in these skin cells. It also significantly inhibited collagen III, interferon gamma-induced protein 10, and intracellular cell adhesion molecule 1. We also studied its effect in regulating genome-wide gene expression. FREO robustly modulated global gene expression. Furthermore, Ingenuity® Pathway Analysis showed that FREO affected many important signaling pathways that are closely related to inflammation, immune response, and tissue remodeling. This study provides the first evidence of the biological activities of FREO in human dermal fibroblasts. Consistent with existing studies in other models, the current study suggests that FREO possesses promising potential to modulate the biological processes of inflammation and tissue remodeling in human skin. Further research into the biological mechanisms of action of FREO and its major active components is recommended.