RESUMO
Nicotine, the most abundant pyridine alkaloid in cultivated tobacco (Nicotiana tabacum L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain function. Nicotine biosynthesis is controlled developmentally and can be induced by abiotic and biotic stressors via a jasmonic acid (JA)-mediated signal transduction mechanism involving members of the APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH) transcription factor (TF) families. AP2/ERF and bHLH TFs work combinatorically to control nicotine biosynthesis and its subsequent accumulation in tobacco leaves. Here, we demonstrate that overexpression of the tobacco NtERF32, NtERF221/ORC1, and NtMYC2a TFs leads to significant increases in nicotine accumulation in T2 transgenic K326 tobacco plants before topping. Up to 9-fold higher nicotine production was achieved in transgenics overexpressing NtERF221/ORC1 under the control of a constitutive GmUBI3 gene promoter compared to wild-type plants. The constitutive 2XCaMV35S promoter and a novel JA-inducible 4XGAG promoter were less effective in driving high-level nicotine formation. Methyljasmonic acid (MeJA) treatment further elevated nicotine production in all transgenic lines. Our results show that targeted manipulation of NtERF221/ORC1 is an effective strategy for elevating leaf nicotine levels in commercial tobacco for use in the preparation of reduced risk tobacco products for smoking replacement therapeutics.
Assuntos
Nicotiana/metabolismo , Nicotina/biossíntese , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Fatores de Transcrição/genética , Acetatos/metabolismo , Alcaloides/biossíntese , Alcaloides/toxicidade , Anabasina/biossíntese , Anabasina/toxicidade , Ciclopentanos/metabolismo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Sequências Hélice-Alça-Hélice/genética , Nicotina/análogos & derivados , Nicotina/economia , Nicotina/toxicidade , Oxilipinas/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/genética , Piridinas/toxicidade , Nicotiana/genética , Produtos do Tabaco/economia , Produtos do Tabaco/toxicidade , Fatores de Transcrição/metabolismoRESUMO
Since seed coating with neonicotinoid insecticides was introduced in the late 1990s, European beekeepers have reported severe colony losses in the period of corn sowing (spring). As a consequence, seed-coating neonicotinoid insecticides that are used worldwide on corn crops have been blamed for honeybee decline. In view of the currently increasing crop production, and also of corn as a renewable energy source, the correct use of these insecticides within sustainable agriculture is a cause of concern. In this paper, a probable--but so far underestimated--route of environmental exposure of honeybees to and intoxication with neonicotinoid insecticides, namely, the atmospheric emission of particulate matter containing the insecticide by drilling machines, has been quantitatively studied. Using optimized analytical procedures, quantitative measurements of both the emitted particulate and the consequent direct contamination of single bees approaching the drilling machine during the foraging activity have been determined. Experimental results show that the environmental release of particles containing neonicotinoids can produce high exposure levels for bees, with lethal effects compatible with colony losses phenomena observed by beekeepers.
Assuntos
Anabasina/toxicidade , Abelhas/efeitos dos fármacos , Exposição Ambiental/análise , Inseticidas/toxicidade , Material Particulado/química , Sementes/química , Zea mays/química , Agricultura , Anabasina/análise , Animais , Guanidinas/análise , Inseticidas/análise , Microscopia Eletrônica de Varredura , Neonicotinoides , Tamanho da Partícula , Tiazóis/análiseRESUMO
The essence of the Druckrey-Küpfmüller equation dtn = constant (where d = daily dose and t = exposure time-to-effect, with n > 1) for chemical carcinogens is that the total dose required to produce the same effect decreases with decreasing exposure levels, even though the exposure times required to produce the same effect increase with decreasing exposure levels. Druckrey and Küpfmüller inferred that if both receptor binding and the effect are irreversible, exposure time would reinforce the effect. The Druckrey-Küpfmüller equation explains why toxicity may occur after prolonged exposure to very low toxicant levels. Recently, similar dose-response characteristics have been established for the toxicity of the neonicotinoid insecticides imidacloprid and thiacloprid to arthropods. This observation is highly relevant for environmental risk assessment. Traditional approaches that consider toxic effects at fixed exposure times are unable to allow extrapolation from measured endpoints to effects that may occur at other times of exposure. Time-to-effect approaches that provide information on the doses and exposure times needed to produce toxic effects on tested organisms are required for prediction of toxic effects for any combination of concentration and time in the environment.
Assuntos
Anabasina/toxicidade , Artrópodes/efeitos dos fármacos , Carcinógenos Ambientais/toxicidade , Inseticidas/toxicidade , Animais , Carcinógenos Ambientais/administração & dosagem , Relação Dose-Resposta a Droga , Poluentes Ambientais/administração & dosagem , Poluentes Ambientais/toxicidade , Imidazóis/administração & dosagem , Imidazóis/toxicidade , Inseticidas/administração & dosagem , Neonicotinoides , Nitrocompostos/administração & dosagem , Nitrocompostos/toxicidade , Ligação Proteica , Piridinas/administração & dosagem , Piridinas/toxicidade , Medição de Risco/métodos , Tiazinas/administração & dosagem , Tiazinas/toxicidade , Fatores de TempoRESUMO
Bombus terrestris bumblebees are important pollinators of wild flowers, and in modern agriculture they are used to guarantee pollination of vegetables and fruits. In the field it is likely that worker bees are exposed to pesticides during foraging. To date, several tests exist to assess lethal and sublethal side-effects of pesticides on bee survival, growth/development and reproduction. Within the context of ecotoxicology and insect physiology, we report the development of a new bioassay to assess the impact of sublethal concentrations on the bumblebee foraging behavior under laboratory conditions. In brief, the experimental setup of this behavior test consists of two artificial nests connected with a tube of about 20 cm and use of queenless micro-colonies of 5 workers. In one nest the worker bees constructed brood, and in the other food (sugar and pollen) was provided. Before exposure, the worker bees were allowed a training to forage for untreated food; afterwards this was replaced by treated food. Using this setup we investigated the effects of sublethal concentrations of the neonicotinoid insecticide imidacloprid, known to negatively affect the foraging behavior of bees. For comparison within the family of neonicotinoid insecticides, we also tested different concentrations of two other neonicotinoids: thiamethoxam and thiacloprid, in the laboratory with the new bioassay. Finally to evaluate the new bioassay, we also tested sublethal concentrations of imidacloprid in the greenhouse with use of queenright colonies of B. terrestris, and here worker bees needed to forage/fly for food that was placed at a distance of 3 m from their hives. In general, the experiments showed that concentrations that may be considered safe for bumblebees can have a negative influence on their foraging behavior. Therefore it is recommended that behavior tests should be included in risk assessment tests for highly toxic pesticides because impairment of the foraging behavior can result in a decreased pollination, lower reproduction and finally in colony mortality due to a lack of food.