Synthesis and turnover of nitrate reductase in corn roots

The induction and reinduction of nitrate reductase in root tip or mature root sections show essentially a similar pattern: a lag, a period of rapid increase in enzyme activity and finally a period of relatively minor change. Both inductions are sensitive to 6-methylpurine and cycloheximide. Kinetic studies with 6-methylpurine suggest that the half-life of the messenger RNA for nitrate reductase in both sections is about 20 minutes. The rate of decay of nitrate reductase activity induced by transfer to a nitrate-free medium is slower in root tips (t½ = 3 hours) than in mature root sections (t½ = 2 hours). The enzyme from mature root sections is also less stable to mild heat treatments (27 C; 40 C) than the enzyme from root tip sections. The results indicate that factors regulating enzyme turnover show important changes as root cells mature and may be significant in determining steady state levels of the enzyme.     

Ammonium and amino acids as regulators of nitrate reductase in corn roots

When amino acids or ammonia are added to plant systems, the effects on the development of nitrate-dependent nitrate reductase activity are variable. In addition, amino acids added singly or as casein hydrolysate may not support a normal growth. A physiologically correct mixture of amino acids, one similar in composition to amino acids released by the endosperm, has been shown to support normal growth and protein synthesis in corn (Zea mays) embryos. In this investigation, we have used the mixture of corn amino acids to determine whether amino acids have an effect on the appearance or disappearance of nitrate reductase activity. The results show that these amino acids partially inhibit the induction of nitrate reductase in corn roots. The effect is more pronounced in mature root than in root tip sections. When glutamine and asparagine are included along with the "corn amino acid mixture," the inhibition is more severe. Amino acids or amino acid analogues added singly to the induction medium have a similar effect: i.e. when the induction of nitrate reductase is inhibited in the root tips (lysine, canavanine, azaserine, azetidine-2-carboxylic acid, dl-4-azaleucine, asparagine, and glutamine), that inhibition is more severe in mature root sections. Arginine enhanced the recovery of nitrate reductase in root tips but inhibited it in mature root sections. The effect of the amino acids is apparently on some phase of the induction processes (i.e. the uptake or distribution of nitrate or a direct effect on the synthesis of the enzyme) and not on the turnover of the enzyme.     

Action of Inhibitors of Ammonia Assimilation on Amino Acid Metabolism in Hordeum vulgare L. (cv Golden Promise)

Barley (Hordeum vulgare L. cv Golden Promise) plants were grown in a continuous culture system in which the root and shoot ammonia and amino acid levels were constant over a 6-hour experimental period. Methionine sulfoximine (MSO), 1 millimolarity when added to the culture medium, caused a total inactivation of root glutamine synthetase with little effect on the shoot enzyme. Root ammonia levels increased and glutamine levels decreased, irrespective of whether the plants were grown in 1 millimolar nitrate or 1 millimolar ammonia. Levels of glutamate, aspartate, serine, threonine, and asparagine all increased. There was little alteration in the amino acid and ammonia levels in the shoot, suggesting that MSO is not rapidly transported.

The addition of azaserine (25 micrograms per milliliter) to nitrate-grown plants caused a rapid increase in root ammonia, glutamine, and serine levels with a corresponding decrease in glutamate, aspartate, and alanine. Glutamine levels also increased in the shoot.

The in vivo effect of MSO and azaserine was as would be predicted by their known in vitro inhibitory action if the glutamine synthetase/glutamate synthase pathway of ammonia assimilation was in operation.

     

Enzymes of asparagine synthesis in maize roots

An asparagine synthetase which is active with either glutamine or NH ₄ ⁺ has been found in maize (Zea mays L.) roots. Unlike the enzyme obtained from legume cotyledons, the maize-root enzyme is only slightly more efficient with glutamine (Km, 1.0 mM) than with NH ₄ ⁺ (Km, 2.0–3.0 mM). The activity of this enzyme is higher in the mature root than in the root-tip region, i.e. root cells develop a capacity to make asparagine from glutamine or NH ₄ ⁺ as they mature. -Cyanoalanine synthetase is also present in maize roots. The apparent Km for cysteine is 2.6 mM and for cyanide is 0.57 mM. The enzyme is more active in the root tip than in mature root tissue. Thus, if asparagine were made in the root tip, the cyanide pathway could represent the mechanism of synthesis. It is our contention, however, that this potential is not realized under normal conditions because ¹⁴C-experiments performed previously have indicated a limited availability of both CN and cysteine in the maize root.     

Effect of glucose on the induction of nitrate reductase in corn roots

In Zea mays L., addition of glucose to the induction medium has no effect on the induction of nitrate reductase during the initial 3 hours either in root tips (0-10 mm) or mature root sections (25-35 mm). With longer times, higher levels of enzyme activity are recovered from both root segments when glucose is present in the incubation medium. The induction in root tips is saturated by 10 mm NO(3) (-). Higher concentrations of NO(3) (-) are required for saturation in mature root sections. The response to glucose is seen over a wide range of external NO(3) (-) concentrations.Nitrate reductase activity is lost rapidly when nitrate is withdrawn from the induction medium. Additions of glucose do not prevent this loss. Additions of glucose have no effect on total uptake of NO(3) (-) by the root segments but they increase the anaerobic NO(2) (-) production in both root tips and mature root segments. This latter measurement is considered to be an estimate of an active NO(3) (-) pool in the cytoplasm. Thus the results show that glucose alters the distribution of NO(3) (-) within the root sections. This may be an important factor in controlling the in vivo stability of the enzyme or its rate of synthesis.     

Negative regulation of asparagine synthetase in the leaves of maize seedlings by light, benzyladenine and glucose

Asparagine synthetase (EC 6.3.5.4) activity was increased 4- and 8-fold when maize ( Zea mays L.) seedlings were kept in darkness for 24 h and 7 days, respectively; this increase was abolished by cycloheximide. Irradiation of the dark adapted seedlings with a pulse of red light resulted in a 4-fold decrease of the enzyme activity within 48 h, which was raised again following a far-red light pulse. Co-action of light and benzyladenine, reported for the light-inducible enzymes, was proved to hold also for the light-repressible asparagine synthetase. The induction of asparagine synthetase activity in the dark is abolished by glucose, suggesting the possible involvement of the enzyme in the contrae of metabolic fluxes of –carbon and nitrogen through assimilatory pathways.     

Induction of asparagine synthetase during lymphocyte activation by phytohemagglutinin

Asparagine synthetase was increased in cultured mouse spleen lymphocytes after stimulation by phytohemagglutinin. After a lag period of about 24h, the enzyme activity level rose sharply by 48h, reached its maximum at 72h, and decreased thereafter. The time course of the change in the enzyme activity was similar to that of the change in the rate of DNA synthesis. From the results that there was no increase of the activity of asparagine synthetase at the time induction of ornithine decarboxylase would occur (6h), it seems unlikely that asparagine synthesized in the cells contributes to the enhancement of ornithine decarboxylase during the activation of lymphocytes. The increase of asparagine synthetase activity was inhibited by cycloheximide and somewhat by actinomycin D, suggesting de novo enzyme synthesis during the stimulation.     

Evidence for cycloheximide acting as a glutamine analogue in plant tissue.

In growing maize root tissue [14C]asparagine formation in inhibited and [14C]glutamine accumulation stimulated by treatment with cycloheximide or glutamine analogs such as azaserine. In contrast, puromycin enhances the accumulation of [14C]asparagine but not [14C]glutamine. Cycloheximide and puromycin alone inhibit protein synthesis. This is interpreted to mean that the alteration in amide metabolism following cycloheximide treatment is a direct result of the antibiotic acting as a glutamine analog. While cycloheximide is often the cytoplasmic protein synthesis inhibitor of choice due to its potency and rapid action, its assumed specificity of action of eukaryotes is doubtful.     

Effect of l-Canavanine on Nitrate Reductase in Corn Roots

l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 μm canavanine was effective in root tips whereas only 50 μm was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO₃ resulted in a loss of NRA. NO₃⁻ protected nitrate reductase from this inactivation in both root tip and mature root sections.     

Cyanide as an asparagine precursor in corn roots

K¹⁴CN is efficiently converted to asparagine in corn roots with asparagine accounting for 26% of the total radioactivity after 2 hr. Additions of glucose, cysteine or serine do not affect the reaction. Cysteine-¹⁴C(U) is normally a poor precursor of asparagine, but in the presence of 10⁻⁶ M KCN becomes a significant source. Cyanide does not promote the incorporation of serine-¹⁴C(U) or acetate-2-¹⁴C into asparagine. The antibiotic cycloheximide is a potent inhibitor of asparagine formation in the root tips when acetate-2-¹⁴C or aspartate-¹⁴C(U) serve as precursors. However, when K¹⁴CN is the precursor it is without effect. The results, therefore, show that cyanide is a potential asparagine precursor in maize root tips and suggest that normally the availability of cyanide and the synthesis of cysteine from serine are major rate limiting reactions in this pathway.     

Elevated levels of asparagine synthetase activity in physiologically and genetically derepressed Chinese hamster ovary cells are due to increased rates of enzyme synthesis

The activity of asparagine synthetase in Chinese hamster ovary (CHO) cells is increased in response to asparagine deprivation or decreased aminoacylation of several tRNAs (Andrulis, I. L., Hatfield, G. W., and Arfin, S. M. (1979) J. Biol. Chem. 254, 10629-10633). CHO cells resistant to beta-aspartylhydroxamate have up to 5-fold higher levels of asparagine synthetase than the parental line (Gantt, J. S., Chiang, C. S., Hatfield, G. W., and Arfin, S. M. (1980) J. Biol. Chem. 255, 4808-4813). We have investigated the basis for these differences in enzyme activity by combined radiochemical and immunological techniques. The asparagine synthetase of beef pancreas was purified to apparent homogeneity. Antibodies raised against the purified protein cross-react with the asparagine synthetase of CHO cells. Immunotitrations show that the amount of enzyme protein in physiologically or genetically derepressed CHO strains is proportional to the level of enzyme activity. Measurement of the relative rates of asparagine synthetase synthesis by pulse-labeling experiments demonstrate that the difference in the number of asparagine synthetase molecules is closely correlated with the rate of enzyme synthesis. In contrast, the half-life of asparagine synthetase in wild type cells and in physiologically or genetically derepressed cells is very similar. It appears that the increased levels of asparagine synthetase can be attributed solely to an increased rate of enzyme synthesis.     

Transport of amino acids to the maize root

When 5-mm maize root tips were excised and placed in an inorganic salts solution for 6 hours, there was a loss of alcohol-insoluble nitrogen. The levels of threonine, proline, valine, isoleucine, leucine, tyrosine, phenylalanine, and lysine in the alcohol soluble fraction were severely reduced, whereas those of glutamate, aspartate, ornithine, and alanine were scarcely affected. There was a 4-fold increase in the level of γ-aminobutyrate. Those amino acids whose synthesis appeared to be deficient in excised root tips also showed poor incorporation of acetate carbon. In addition, the results show that asparagine and the amino acids of the neutral and basic fraction were preferentially transported to the root tip region. The results therefore suggest that the synthesis of certain amino acids in the root tip region is restricted, and that this requirement for amino acids in the growing region could regulate the flow of amino acids to the root tip.     

Particulate glucan synthetase activity: generation and inactivation after treatments with indoleacetic acid and cycloheximide

Growing regions from epicotyls of Pisum sativum L. var Alaska contain a particulate enzyme which transfers glucose from guanosine diphosphate glucose to alkali-soluble and -insoluble products (glucan synthetase activity). When the epicotyl is decapitated to remove the source of natural hormone, the tissue below ceases growth and loses synthetase activity as well as the capacity to continue forming cellulose in vivo. If indoleacetic acid (IAA) is added to the cut apex, massive amounts of cellulose are deposited in the next few days. Particulate glucan synthetase activity is either maintained or greatly increased depending on whether endogenous activity levels are relatively high or low at the time of hormone addition. These effects appear to be due in part to IAA-dependent generation of a protein essential for synthetase activity since they are severely inhibited by concentrations of cycloheximide which are effective at preventing protein synthesis. Nevertheless, the addition of cycloheximide alone to the epicotyl reduces the rate of disappearance of synthetase activity, i.e., a protective effect. Also, a soluble thermolabile component is present in the aging epicotyl which promotes loss of synthetase activity when added to the particulate enzyme in vitro. Accordingly, turnover of pea glucan synthetase activity may be controlled in part by an inactivating protein which is itself subject to turnover.     

Tissue specific localization of root infection by fungal pathogens: role of root border cells

When roots of pea seedlings were inoculated uniformly with spores of Nectria haematocca or other pea pathogenic fungi, more than 90% developed lesions in the region of elongation within 3 days. More mature regions of most roots as well as the tip showed no visible signs of infection. Yet, microscopic observation revealed that 'mantles,' comprised of fungal hyphae intermeshed with populations of border cells, covered the tips of most roots. After physical detachment of the mantle, the underlying tip of most roots was found to be free of infection. Mantle-covered root tips did not respond to invasion of their border cells by activation of known defense genes unless there was invasion of the tip itself, as revealed by the presence of a lesion. Concomitant with the activation of defense genes was the induction of a cell-wall degrading enzyme whose expression is a marker for renewed production of border cells. Mantle formation did not occur in response to nonpathogens. The data are consistent with the hypothesis that border cells serve as a host-specific 'decoy' that protects root meristems by inhibiting fungal infection of the root tip.     

Methotrexate stimulation of asparagine synthetase activity in rat liver

Methotrexate was found to stimulate asparagine synthetase activity in vivo by approximately six-fold in rat liver. The maximum effect of methotrexate on hepatic asparagine synthetase activity was observed sixteen hours after intraperitoneal injection of the drug. Cycloheximide, like methotrexate, is a protein synthesis inhibitor and was used to determine that asparagine synthetase activity was not preferentially stimulated under stress. As expected, hepatic asparagine synthetase activity falls markedly with the decreased protein synthesis caused by injection of cycloheximide. It is proposed that methotrexate inhibits serine-dependent glycine biosyn-thesis by decreasing the concentration of tetrahydrofolate for serine hydroxymethyltransferase. This leads to a stimulation of asparagine synthetase to provide nitrogen for asparagine-dependent glycine synthesis. This may provide an explanation of the observed chemotherapeutic synergism between asparaginase and methotrexate treatment.     

Studies of the kinetics of oxidation of cytochrome c by cytochrome c oxidase: comparison of the reactions of purified and membrane-bound oxidase

l-Asparaginase (EC 3.5.1.1.) activity has been detected in crude extracts of Lupinus arboreus young leaves, root tips, flower buds, and developing seeds. The enzyme was also present in Lupinus angustifolius root tips, developing nodules, and developing seeds. The asparaginase from each of these tissues had the same electrophoretic mobility on polyacrylamide gels and a K_m of 6–8 mm for asparagine. In extracts other than those of the developing seeds, asparaginase activity was dependent upon the inclusion of K⁺ ion and a sulfhydryl protectant in the extraction buffer. No asparaginase activity was detected in mature leaves, in the plant fraction of nodules that were fixing nitrogen, nor in root tissue further than 1.5 cm from the root tip. Asparaginase has been purified 326- and 230-fold from L. arboreus and L. angustifolius developing seeds, respectively. A molecular weight of 75,000 was obtained by gel filtration. An apparent K_m of 6.6 and 7.0 mm for asparagine was determined for the purified L. arboreus and L. angustifolius asparaginases, respectively. Of the amides, nitriles, and hydroxamates examined, the L. arboreus enzyme hydrolyzed only l-asparagine and dl-aspartyl hydroxamate. This same enzyme was inhibited by d-asparagine, 5-diazo-4-oxo-l-norvaline, dl-aspartyl hydroxamate, d-and l-aspartate, 3-cyano-l-alanine, glycine, and cysteine. Glutamine, glutamine analogs, and a number of other amino acids, amides and amines did not inhibit the L. arboreus asparaginase.     

Calcium, asparagine and cAMP are required for ornithine decarboxylase activation in intact Chinese hamster ovary cells.

Asparagine specifically activated ornithine decarboxylase activity 5–7 fold by 7–8 h in confluent cultures maintained with a salts/glucose medium. When dibutyryl cAMP was added with asparagine, a 40–50 fold stimulation of ornithine decarboxylase activity was produced. Ornithine decarboxylase activation in the salts/glucose medium was not sensitive to actinomycin D. Omission of Ca⁺⁺ and Mg⁺⁺ from the medium abolished the ability of asparagine and/or dibutyryl cAMP to stimulate enzyme activity. Calcium was essential for the asparagine and dibutyryl cAMP mediated stimulation of ornithine decarboxylase activity.     

Contrasted Responses to Root Hypoxia in Tomato Fruit at Two Stages of Development

The biochemical consequences of root hypoxia have been documented in many sink organs, but not extensively in fruit. Therefore, in the present study, the response to root hypoxia in tomato fruit (Solanum lycopersicum L.) was investigated at two developmental stages, during the cell division and the cell expansion phases. Our results showed that in dividing fruit, root hypoxia caused an exhaustion of carbon reserves and proteins. However, ammonium and major amino acids (glutamine, asparagine and γ–aminobutyric acid (GABA)) significantly accumulated. In expanding fruit, root hypoxia had no effect on soluble sugar, protein and glutamine contents, whereas starch content was significantly decreased, and asparagine and GABA contents slightly increased. Metabolite contents were well correlated with activities of the corresponding metabolising enzymes. Contrary to nitrogen metabolising enzymes (glutamine synthetase, asparagine synthetase and glutamate decraboxylase), the activities of enzymes involved in sugar metabolism (invertase, sucrose synthase, sucrose phosphate synthase and ADP glucose pyrophosphorylase) were significantly reduced by root hypoxia, in diving fruit. In expanding fruit, only a slight decrease in ADP glucose pyrophosphorylase and an increase in asparagine synthetase and glutamate decarboxylase activities were observed. Taken together, the present data revealed that the effects of root hypoxia are more pronounced in the youngest fruits as it is probably controlled by the relative sink strength of the fruit and by the global disturbance in plant functioning.