RESUMO
Phylloquinone (Vitamin K(1)) is an essential component of the photosynthetic electron transfer. As isochorismate is required for the biosynthesis of Vitamin K(1), isochorismate synthase (ICS) activity is expected to be present in all green plants. In bacteria salicylic acid (SA) is synthesized via a two step pathway involving ICS and isochorismate pyruvate lyase (IPL). The effect of the introduction in tobacco plants of the bacterial ICS and IPL genes on the endogenous isochorismate pathway was investigated. Transgenic tobacco plants in which IPL was targeted to the chloroplast suffered severe growth retardation and had low Vitamin K(1) content. Probably because isochorismate was channeled towards SA production, the plants were no longer able to produce normal levels of Vitamin K(1). Transgenic tobacco plants in which the bacterial ICS was present in the chloroplast showed higher Vitamin K(1) contents than wild type plants.
Assuntos
Vias Biossintéticas/genética , Carbono-Oxigênio Liases/genética , Transferases Intramoleculares/genética , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Ácido Salicílico/metabolismo , Vitamina K 1/metabolismo , Proteínas de Bactérias/genética , Carbono-Oxigênio Liases/metabolismo , Ácido Corísmico/metabolismo , Ácidos Cicloexanocarboxílicos/metabolismo , Cicloexenos/metabolismo , Transferases Intramoleculares/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/genética , Nicotiana/metabolismo , Vitamina K 1/isolamento & purificaçãoRESUMO
A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.
Assuntos
Cannabis/enzimologia , DNA Complementar/genética , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Acil Coenzima A/metabolismo , Aciltransferases/genética , Sequência de Aminoácidos , Sequência de Bases , Canabinoides/biossíntese , Cannabis/genética , Cannabis/metabolismo , Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Malonil Coenzima A/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Homologia de Sequência de AminoácidosRESUMO
The hypersensitive interaction between Tobacco mosaic virus (TMV) and tobacco results in accumulation of salicylic acid (SA), defense gene expression, and development of systemic acquired resistance (SAR) in uninfected leaves. The plant hormones SA and ethylene have been implicated in SAR. From a study with ethylene-insensitive (Tetr) tobacco, we concluded that ethylene perception is required to generate the systemic signal molecules in TMV-infected leaves that trigger SA accumulation, defense gene expression, and SAR development in uninfected leaves. Ethylene perception was not required for the responses of the plant to the systemic signal that leads to SAR development.
Assuntos
Etilenos/farmacologia , Nicotiana/efeitos dos fármacos , Nicotiana/fisiologia , Transdução de Sinais/efeitos dos fármacos , Vírus do Mosaico do Tabaco/imunologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Nicotiana/genética , Nicotiana/virologia , TransplantesRESUMO
Salicylic acid (SA) is a signalling compound in plants which is able to induce systemic acquired resistance. In the analysis of SA in plant tissues, the extraction recovery is often very low and variable. This is mainly caused by sublimation of SA, especially during evaporation of organic solvents. Techniques have been designed in order to overcome this problem. In the first part of the extraction procedure, sublimation of SA was prevented by addition of 0.2 M sodium hydroxide. At a later stage of the extraction procedure, sublimation of SA during solvent evaporation was controlled by the addition of a small amount of HPLC eluent. In this way, recoveries in the range of 71-91% for free SA and 65-79% for acid-hydrolysed SA were obtained. Recoveries could be further optimised by the use of an internal standard to correct for volume changes after the addition of the HPLC eluent.
