RESUMO
Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation.
Assuntos
Metais Pesados/metabolismo , Fitoquelatinas/metabolismo , Aminoaciltransferases/química , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cádmio/metabolismo , Cádmio/toxicidade , Domínio Catalítico/genética , Quelantes/metabolismo , Evolução Molecular Direcionada , Recuperação e Remediação Ambiental , Intoxicação por Metais Pesados , Engenharia Metabólica , Modelos Moleculares , Mostardeira/efeitos dos fármacos , Mostardeira/genética , Mostardeira/metabolismo , Mutagênese , Fitoquelatinas/química , Fitoquelatinas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Intoxicação/metabolismo , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Petunia hybrida genetic transformation continues to be a valuable tool for genetic research into biochemical pathways and gene expression, as well as generating commercial products with varying floral colors. In this chapter, we describe a simple and reproducible genetic transformation protocol for generating transgenic petunia plants harboring a gene of interest and selectable marker. The system utilizes Agrobacterium tumefaciens for transgene integration with plant recovery via shoot organogenesis from leaf explant material. Selection for transgenic plants is achieved using the bar gene conferring resistance to glufosinate or nptII gene for resistance to kanamycin. Transformation efficiencies of around 10% are achievable with shoots being recovered about 8 wk after transgene insertion and rooted plants transferred to the greenhouse about twelve weeks after inoculation.
Assuntos
Agrobacterium tumefaciens/genética , Petunia/genética , Transformação Genética , Agrobacterium tumefaciens/citologia , Anti-Infecciosos , Técnicas de Cultura de Células , Técnicas de Cocultura , Meios de Cultura , Germinação , Petunia/anatomia & histologia , Petunia/fisiologia , Reguladores de Crescimento de Plantas , Brotos de Planta/anatomia & histologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regeneração , Solo , Esterilização , Técnicas de Cultura de Tecidos , TransgenesRESUMO
Limitations in transformation capability can be a significant barrier in making advances in our understanding of gene function through the use of transgenics. To this end we have developed both tissue culture and non-tissue culture-based methodologies for the production of transgenic roots on wild-type shoots (composite plants). Composite plants are generated by inoculating wild-type shoots with Agrobacterium rhizogenes, which subsequently induces the formation of transgenic roots. The composite plant system allows for "in root" testing of transgenes in the context of a complete plant and can be analyzed in a variety of gene function analyses and plant-microbe interaction studies. In this chapter we provide a tissue culture-based composite plant generation system for Arabidopsis and a non-tissue culture based-method for producing composite plants on a variety of dicotyledonous plant species. Composite plants generated using these methods can be treated like "normal plants," planted in soil and grown in greenhouses or in growth chambers. These methods have been shown to work efficiently for many different species of plants including several that are recalcitrant to transformation.
