RESUMEN

Control of messenger RNA (mRNA) stability serves as an important mechanism for regulating gene expression. Analysis of Arabidopsis mutants that overaccumulate soluble methionine (Met) revealed that the gene for cystathionine gamma-synthase (CGS), the key enzyme in Met biosynthesis, is regulated at the level of mRNA stability. Transfection experiments with wild-type and mutant forms of the CGS gene suggest that an amino acid sequence encoded by the first exon of CGS acts in cis to destabilize its own mRNA in a process that is activated by Met or one of its metabolites.

Asunto(s)

Arabidopsis/enzimología , Liasas de Carbono-Oxígeno/genética , Regulación de la Expresión Génica de las Plantas , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Liasas de Carbono-Oxígeno/química , Liasas de Carbono-Oxígeno/metabolismo , Exones , Regulación Enzimológica de la Expresión Génica , Genes de Plantas , Genes Reporteros , Cinética , Metionina/metabolismo , Datos de Secuencia Molecular , Mutación , ARN Mensajero/genética , Alineación de Secuencia , Transcripción Genética , Transfección

RESUMEN

The specific growth rate of stable Bacillus subtilis L-forms was slower (mu = 0.127) than the cell-walled form (mu = 0.219) when measured by optical density (O.D.). However, the L-form growth rate increased (mu = 0.288) when determined by viable cell counts. L-forms of B. subtilis appear to enter a phase of rapid cell division, followed by a period of cell enlargement which is associated with an increase in the number of cells with vacuoles and granules. Thus, maximum viable L-form numbers and DNA content were attained at approx. 30h, before maximum protein content was achieved (46h) and before maximum O.D. was observed at 71 h. Measurements showed that L-form cell size increased even after cell division had stopped. O.D. was therefore inaccurate for assessment of L-form growth. L-forms were sensitive to osmotic shock and unlike the cell-walled organisms from which they were derived, were resistant to penicillin, indicating a loss of peptidoglycan. The L-forms were similar to cell-walled forms in that antibiotic(s) and proteases were produced.

Asunto(s)

Bacillus subtilis/crecimiento & desarrollo , Formas L/crecimiento & desarrollo , Antibacterianos/biosíntesis , Bacillus subtilis/fisiología , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/análisis , Recuento de Colonia Microbiana , ADN Bacteriano/análisis , Cinética , Formas L/fisiología , Muramidasa/farmacología , Penicilinas/farmacología , Vacuolas/ultraestructura , Agua/farmacología

RESUMEN

Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase (14)C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent K(m) (M6P) was 15.8 millimolar, but the apparent K(m) (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg(2+), Ca(2+), Zn(2+), and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPH-dependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.

RESUMEN

We have used (13)C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of (13)C between carbons atoms 1 and 6 of [1-(13)C] or [6-(13)C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the (13)C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the (13)C sugars were supplied in vivo to the intact plant. The (13)C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of (13)C in all glucosyl carbons was quantified by (13)C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background (13)C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate interconversion in the amyloplast could not be detected. These data seriously weaken the argument for the selective uptake of triose phosphates by the amyloplast as part of the pathway of starch biosynthesis from sucrose in plant storage tissues. Instead, we suggest that a hexose phosphate such as glucose 1-phosphate, glucose 6-phosphate, or fructose 6-phosphate is the most likely candidate for entry into the amyloplast. A pathway of starch biosynthesis is presented, which is consistent with our data and with the current information on the intracellular distribution of enzymes in plant storage tissues.

RESUMEN

The aim of this work was to discover which compound(s) cross the amyloplast envelope to supply the carbon for starch synthesis in grains of Triticum aestivum L. Amyloplasts were isolated, on a continuous gradient of Nycodenz, from lysates of protoplasts of endosperm of developing grains, and then incubated in solutions of (14)C-labelled: glucose, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glycerol 3-phosphate. Only glucose 1-phosphate gave appreciable labelling of starch that was dependent upon the integrity of the amyloplasts. Incorporation into starch was linear with respect to time for 2 h. At the end of the incubations, 98% of the (14)C in the soluble fraction of the incubation mixture was recovered as [(14)C]glucose 1-phosphate. Thus it is unlikely that the added [(14)C glucose 1-phosphate was extensively metabolized prior to uptake by the amyloplasts. It is argued that the behaviour of the isolated amyloplasts, and previously published data on the labelling of starch by [(13)C]glucose, are consistent with the view that in wheat grains it is a C-6, not a C-3, compound that enters the amyloplast to provide the carbon for starch synthesis.