RESUMO
Jatropha curcas contains seeds with a high oil content, suitable for biodiesel production. After oil extraction, the remaining mass can be a rich source of enzymes. However, data from the literature describing physicochemical characteristics for a monomeric esterase from the J. curcas seed did not fit the electrostatic catapult model for esterases/lipases. We decided to reevaluate this J. curcas esterase and extend its characterization to check this apparent discrepancy and gain insights into the enzyme's potential as a biocatalyst. After anion exchange chromatography and two-dimensional gel electrophoresis, we identified the enzyme as belonging to the dienelactone hydrolase family, characterized by a cysteine as the nucleophile in the catalytic triad. The enzyme displayed a basic optimum hydrolysis pH of 9.0 and an acidic pI range, in contrast to literature data, making it well in line with the electrostatic catapult model. Furthermore, the enzyme showed low hydrolysis activity in an organic solvent-containing medium (isopropanol, acetonitrile, and ethanol), which reverted when recovering in an aqueous reaction mixture. This enzyme can be a valuable tool for hydrolysis reactions of short-chain esters, useful for pharmaceutical intermediates synthesis, due to both its high hydrolytic rate in basic pH and its stability in an organic solvent.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Jatropha/enzimologia , Modelos Moleculares , Eletricidade Estática , Sequência de Aminoácidos , Análise de Variância , Hidrolases de Éster Carboxílico/química , Domínio Catalítico , Cátions Bivalentes/farmacologia , Esterases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Ponto Isoelétrico , Proteólise/efeitos dos fármacos , Proteômica , Solventes , Estereoisomerismo , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
Aluminum (Al) toxicity has been recognized to be a main limiting factor of crop productivity in acid soil. Al interacts with cell walls disrupting the functions of the plasma membrane and is associated with oxidative damage and mitochondrial dysfunction. Jatropha curcas L. (J. curcas) is a drought resistant plant, widely distributed around the world, with great economic and medicinal importance. Here we investigated the effects of Al on J. curcas mitochondrial function and cell viability, analyzing mitochondrial respiration, phenolic compounds, reducing sugars and cell viability in cultured J. curcas cells. The results showed that at 70⯵M, Al limited mitochondrial respiration by inhibiting the alternative oxidase (AOX) pathway in the respiratory chain. An increased concentration of reducing sugars and reduced concentration of intracellular phenolic compounds was observed during respiratory inhibition. After inhibition, a time-dependent upregulation of AOX mRNA was observed followed by restoration of respiratory activity and reducing sugar concentrations. Cultured J. curcas cells were very resistant to Al-induced cell death. In addition, at 70⯵M, Al also appeared as an inhibitor of cell wall invertase. In conclusion, Al tolerance in cultured J. curcas cells involves a inhibition of mitochondrial AOX pathway, which seems to start an oxidative burst to induce AOX upregulation, which in turn restores consumption of O2 and substrates. These data provide new insight into the signaling cascades that modulate the Al tolerance mechanism.
Assuntos
Alumínio/farmacologia , Jatropha/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Técnicas de Cultura de Células , Jatropha/enzimologia , Jatropha/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Oxirredução/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Consumo de Oxigênio/efeitos dos fármacos , Proteínas de Plantas/antagonistas & inibidoresRESUMO
Ascorbate peroxidase (APX) plays a central role in the ascorbate-glutathione cycle and is a key enzyme in cellular H2O2 me-tabolism. It includes a family of isoenzymes with different character-istics, which are identified in many higher plants. In the present study, we isolated the APX gene from Jatropha curcas L, which is similar with other previously characterized APXs as revealed by alignment and phylogenetic analysis of its deduced amino acid sequence. Real-time qPCR analysis showed that the expression level of JcAPX transcript significantly increased under NaCl stress. Subsequently, to elucidate the contribution of JcAPX to the protection against salt-induced oxi-dative stress, the expression construct p35S: JcAPX was created and transformed into Arabidopsis and transcribed. Under 150-mM NaCl stress, compared with wild type (WT), the overexpression of JcAPX in Arabidopsis increased the germination rate, the number of leaves, and the rosette area. In addition, the transgenic plants had longer roots, higher total chlorophyll content, higher total APX activity, and lower H2O2 content than the WT under NaCl stress conditions. These results suggested that higher APX activity in transgenic lines increases the salt tolerance by enhancing scavenging capacity for reactive oxygen spe-cies under NaCl stress conditions.
Assuntos
Arabidopsis/genética , Ascorbato Peroxidases/genética , Plantas Geneticamente Modificadas/genética , Tolerância ao Sal/genética , Arabidopsis/crescimento & desenvolvimento , Clorofila/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Jatropha/enzimologia , Plantas Geneticamente Modificadas/fisiologia , Tolerância ao Sal/fisiologia , Cloreto de Sódio/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
Superoxide dismutases (SODs) are involved in protecting plants against diverse biotic and abiotic stresses. In the present study, a novel Cu/Zn-SOD gene (JcCu/Zn-SOD) was cloned from Jatropha curcas L. Quantitative reverse transcription-polymerase chain reaction analysis revealed that JcCu/Zn-SOD is constitutively expressed in different tissues of J. curcas and induced under NaCl treatment. To characterize the function of this gene with respect to salt tolerance, the construct p35S:JcCu/Zn-SOD was developed and transformed into Arabidopsis using Agrobacterium-mediated transformation. Compared with wild-type, transgenic plants over-expressing JcCu/Zn-SOD showed enhanced tolerance to salt stress during germination, seedling establishment, and growth in terms of longer root, larger rosette area, and a larger number of leaves in addition to higher SOD activity levels under NaCl stress. In addition, over-expression of JcCu/Zn-SOD resulted in lower monodialdehyde content in transgenic Arabidopsis compared to wild-type plants under the same NaCl stress. Therefore, JcCu/Zn-SOD can increase a plant salt stress tolerance potentially by reducing oxidant injury.
Assuntos
Arabidopsis/enzimologia , Jatropha/enzimologia , Tolerância ao Sal/fisiologia , Superóxido Dismutase/fisiologia , Arabidopsis/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Jatropha/genética , Plantas Geneticamente Modificadas , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/enzimologia , Plantas Tolerantes a Sal/genética , Cloreto de Sódio , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene from J. curcas. IPI is one of the rate limiting enzymes in the biosynthesis of terpenoids, catalyzing the crucial interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Results A full-length JcIPI cDNA consisting of 1355 bp was cloned. It encoded a protein of 305 amino acids. Analysis of deduced amino acid sequence predicted the presence of conserved active sites, metal binding sites and the NUDIX motif, which were consistent with other IPIs. Phylogenetic analysis indicated a significant evolutionary relatedness with Ricinus communis. Southern blot analysis showed the presence of an IPI multigene family in J. curcas. Comparative expression analysis of tissue specific JcIPI demonstrated the highest transcript level in flowers. Abiotic factors could induce the expression of JcIPI. Subcellular distribution showed that JcIPI was localized in chloroplasts. Conclusion This is the first report of cloning and characterization of IPI from J. curcas. Our study will be of significant interest to understanding the regulatory role of IPI in the biosynthesis of terpenoids, although its function still needs further confirmation.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Jatropha/enzimologia , Jatropha/química , Hemiterpenos/genética , Hemiterpenos/metabolismo , Filogenia , RNA/isolamento & purificação , Expressão Gênica , Cloroplastos , Southern Blotting , Clonagem Molecular , DNA Complementar/síntese química , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The vegetation of metal-contaminated soils using non-edible crops can be a safe and economical technique for Cd immobilization and the remediation of contaminated sites. Jatropha (Jatropha curcas L.) exhibits a relative tolerance to heavy metals and potential for biofuel production. The study was performed to monitor the Cd-induced alterations in jatropha plants by X-ray chlorophyll fluorescence. The Cd effects on photosynthetic pigments, the mineral composition of plants, defense enzyme activity and soluble proteins were also studied. Plants were grown for 20days in a nutrient solution with five Cd contents: 5, 10, 20, 30 and 40µmolL(-1); a control with no Cd addition was also monitored. The analysis of the chlorophyll fluorescence spectra allowed detecting alterations caused by Cd toxicity in the jatropha plants. The mineral composition of the plants was affected by the Cd doses; however, the Fe and Mg contents were not significantly reduced, which most likely improved the effects on the contents of the photosynthetic pigments. Because of its relative tolerance to Cd, Jatropha curcas may be a promising species to revegetate Cd-contaminated sites. Considering the long period needed to phytoremediate soils, the combination of remediation with bioenergy production could be an attractive option.