RESUMEN
BACKGROUND AND AIMS: Myo-inositol-1l-phosphate synthase (MIPS) catalyses the conversion of d-glucose 6-phosphate to 1-l-myo-inositol-1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. Inositol phospholipids play a vital role in membrane trafficking and signalling pathways, auxin storage and transport, phytic acid biosynthesis, cell wall biosynthesis and production of stress-related molecules. In the present study, an MIPS cDNA from developing Passiflora edulis f. flavicarpa seeds was characterized and an investigation made into its spatial and differential expression, as well as changes in its transcription during exposure of growing plants to cold and heat stresses. METHODS: The MIPS-encoding gene was isolated by polymerase chain reaction (PCR) methods, and transcript levels were examined using semi-quantitative reverse transcription-PCR (RT-PCR) during seed development and in response to heat and cold stress. In addition, the copy number of the cloned PeMIPS1 gene in the genome of Passiflora edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea was determined by Southern blot analyses. KEY RESULTS: A full-length cDNA clone of the PeMIPS1 from P. edulis was isolated and characterized. Southern blot analyses indicated that the genomic DNA might have diverse sequences of MIPS-encoding genes and one copy of the cloned PeMIPS1 gene in the genomes of P. edulis, P. eichleriana, P. caerulea, P. nitida and P. coccinea. RT-PCR expression analyses revealed the presence of PeMIPS1 transcripts in ovules, pollen grains and leaves, and during the seed developmental stages, where it peaked at 9 d after pollination. The PeMIPS1 gene is differentially regulated under cold and heat stress, presenting a light-responsive transcription. CONCLUSIONS: Experimental data suggest that PeMIPS1 transcription plays an important role in the establishment of developmental programmes and during the response of plants to environmental changes. The PeMIPS1 is differentially transcribed during cold and heat stress, presenting a light response pattern, suggesting that it is important for environmental stress response.
Asunto(s)
Ambiente , Regulación de la Expresión Génica de las Plantas , Mio-Inositol-1-Fosfato Sintasa/genética , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Passiflora/enzimología , Passiflora/genética , Clonación Molecular , Frío , Regulación del Desarrollo de la Expresión Génica , Calor , Luz , Datos de Secuencia Molecular , Filogenia , Semillas/enzimología , Transcripción GenéticaRESUMEN
Inositol plays a role in membrane trafficking and signaling in addition to regulating cellular metabolism and controlling growth. In plants, the myo-inositol-1-phosphate is synthesized from glucose 6-phosphate in a reaction catalyzed by the enzyme myo-inositol-1-phosphate synthase (EC 5.5.1.4). Inositol can be converted into phytic acid (phytate), the most abundant form of phosphate in seeds. The path to phytate has been suggested to proceed via the sequential phosphorylation of inositol phosphates, and/or in part via phosphatidylinositol phosphate. Soybean [Glycine max (L.) Merrill] lines were produced using interfering RNA (RNAi) construct in order to silence the myo-inositol-1-phosphate (GmMIPS1) gene. We have observed an absence of seed development in lines in which the presence of GmMIPS1 transcripts was not detected. In addition, a drastic reduction of phytate (InsP6) content was achieved in transgenic lines (up to 94.5%). Our results demonstrated an important correlation between GmMIPS1 gene expression and seed development.
Asunto(s)
Glycine max/genética , Mio-Inositol-1-Fosfato Sintasa/genética , Ácido Fítico/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Interferencia de ARN , Semillas/crecimiento & desarrollo , Cotiledón/citología , Cotiledón/enzimología , Cotiledón/genética , Microscopía Electrónica de Transmisión , Mio-Inositol-1-Fosfato Sintasa/antagonistas & inhibidores , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/enzimología , Semillas/genética , Glycine max/embriología , Glycine max/enzimologíaAsunto(s)
Eliminación de Gen , Marcación de Gen , Leishmania mexicana/crecimiento & desarrollo , Leishmaniasis Cutánea/parasitología , Mio-Inositol-1-Fosfato Sintasa/genética , Animales , Inositol/metabolismo , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Leishmania mexicana/patogenicidad , Leishmaniasis Cutánea/fisiopatología , Ratones , Datos de Secuencia Molecular , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Reacción en Cadena de la Polimerasa , VirulenciaRESUMEN
The biosynthetic pathway for myo-inositol consist of two enzymatic steps: first, the cycloaldolization of glucose-6P to L-myo-inositol-IP followed by its hydrolysis to form free myo-inositol. The former reaction is catalyzed by myo-inositol-IP synthase (MIPS) while, a phosphatase is responsible for the hydrolysis step. Depending on its degree of purification and storage age, MIPS activity us to be, from partial to fully, dependent on added NAD. Therefore, we decided to study the kinetic properties of the enzyme within the cell, specially its requirements for free NAD. To this purpose, a method was designed for the assay of MIPS-activity in situ, using toluene permeabilized mycelia. MIPS-activity "in situ" was fully displayed in the absence of added NAD; on the contrary, the purified enzyme showed only 33% of that activity displayed when NAD was included in the assay. Thus, it seems that the native enzyme contains tightly bound NAD, instrumental for its activity, and that during purification or storage, the coenzyme is progressively lost, rendering the NAD-dependent enzyme, as was previously envisage. In addition, the in situ assay method for MIP-Synthase was applied to several mutants of N. crassa having the inosphenotype. Our results showed that only in 3 of 14 cases analyzed the phenotype could be clearly associated to the lack of MIP-synthase activity. Indeed most of the mutants analyzed showed significant levels (from 5 to 21%) of MIP-synthase, when compared to the activity shown by the RL-21 WT strain. Finally, all the mutants and WT strains were zymographically analyzed for phosphatase activity and showed close to equal strong reaction levels.