The regulation of glutamate dehydrogenase,nitrite reductase,and nitrate reductase in excised pea roots by nitrite

Both nitrite reductase and nitrate reductase were induced by nitrite, but there were differences in the time course of induction and in the response to different NO₂ ^- concentrations between these enzymes. NH₄ ⁺ depressed the induction of nitrite reductase. NADH₂ dependent glutamate dehydrogenase activity was enhanced by those NO₂-concentrations in the medium at which unmetabolized NO₂ ^- occurred in the roots. NADPH₂ and NAD+ dependent GDh activities were not affected. In vivo modification and (or) in vivo activation were probably responsible for the increase in NADH₂ dependent GDH activity.     

Nitrate uptake and induction of nitrate reductase in excised corn roots

The characteristics of nitrate uptake and induction of nitrate reductase were studied in excised roots of corn (Zea mays L.). Upon initial exposure to nitrate, the low initial rate of nitrate uptake gradually increased until a steady uptake rate was achieved in 1 to 2 hours depending on the NO(3) (-) concentration. The pattern was observed over a wide range (0.2-5 mm) of nitrate concentrations and was independent of the accompanying cation.The nitrate uptake pattern as a function of increasing external nitrate concentrations (0.2-50 mm) followed saturation type kinetics. The reciprocal plot of the data was not linear but hyperbolic, indicating that more than one Km for nitrate uptake can be resolved from the data. This suggests the existence of either one carrier system with changing kinetic constants or the existence of dual uptake systems. The pattern of induction of nitrate reductase was coincident with the pattern of nitrate uptake as a function of time and increasing nitrate concentrations. The rate of induction of nitrate reductase was regulated by the rate of nitrate flux.Washing the roots for 2 hours enhances nitrate uptake by 2.5-fold over the nonwashed tissue. The presence of nitrate in the washing solution leads to further (3.5-fold over control) increases in the rate of nitrate uptake supporting the contention that nitrate plays a specific role in the induction of the inducible nitrate carrier independent of the washing effect.     

Regulation of nitrate reductase in excised barley roots

When excised barley roots (Hordeum distichum L.) are appropriately pretreated, the level of nitrate reductase in the roots increases upon exposure to nitrate. Relatively low levels of nitrate (10 mum) gave maximum induction of nitrate reductase. This increase was inhibited by inhibitors of protein and RNA synthesis, indicating that de novo protein synthesis is probably involved. Induction of nitrate reductase by nitrate is partially prevented by the inclusion of ammonium, an eventual product of nitrate reduction, in the incubation medium. Under the experimental conditions used, ammonium did not inhibit the uptake of nitrate by excised barley roots. It is concluded, therefore, that ammonium, or a product of ammonium metabolism, has a direct effect on the synthesis of nitrate reductase in this tissue.     

The inhibition of nitrate reductase induction by spermine in excised radish cotyledons

The induction of nitrate reductase in excised cotyledons of radish seedlings was inhibited by the polyamines, spermidine and spennine, in light and dark, but putrescine had no effect. Spermine had no effect on the uptake of nitrate or the stability of the enzyme, but inhibited the synthesis of the enzyme.     

The effect of chloride on nitrate reductase level,on anaerobic nitrite production,and on nitrate content in excisedPisum sativum L. roots

The effect was studied of chloride ions, added in the form of different salts, on nitrate reductase (NR) level in excised pea roots, on anaerobic nitrite production in an assay medium lacking both nitrate and n-propanol, on nitrate content in the roots, and on in vivo NR activity determined in an assay medium containing 5% n-propanol. The presence of Cl in nitrate containing nutrient solutions resulted in lower NR levels, however counterions supplied together with Cl tended to modify slightly this general trend. The negative effect of Cl ions was also apparent, when Cl ions were applied before nitrate ions. Anaerobic nitrite production in the medium lacking both nitrate and n-propanol was not influenced by chloride ions. Nitrate content in the roots was reduced in the presence of chloride both at 3 mM and 15 mM NO₃ in nutrient solutions; however, at 16 mM NO₃, nitrate content in the roots exoeeded even in the presence of 15 mM Cl nitrate content in those root segments which were cultivated in a nutrient solution with 6 mM nitrate, which is the concentration at which NR reaches the level of saturation in excised pea roots. The results obtained suggest that a special induction nitrate pool exists in plant cells besides the storage and metabolic nitrate pools.     

Inhibition of nitrate reductase induction by canavanine in maize roots

The induction of nitrate reductase activity in maize root tips was inhibited by canavanine and the inhibition increased with increasing concentration of canavanine between 0·1 and 1 mM. Addition of canavanine to the induced enzyme had little effect on the disappearance of the enzyme when nitrate was removed, and it is likely that the canavanine reduces the activity of the nitrate reductase by inhibiting its synthesis rather than by accelerating its breakdown.     

The influence of pretreatment with different cations on anaerobic nitrite production by excisedPisum sativum roots

The influence of pretreatment with some cations on anaerobic nitrite production (in an assay medium lacking nitrate) by excised primary roots of pea (Pisum sativum L., ov. Raman), detached from six-day-old seedlings germinated in distilled water, was investigated. When the excised roots were precultivated in one-salt-solutions of KNO3, then these roots produced at 9 mM and 15 mM NO3- concentrations under anaerobic conditions significantly more NO2-, than those precultivated in a nutrient solution containing besides K+ ions also Ca2+ and Mg2+ ions, and they produced nitrite for a longer time. The KNO3 dependent increase in anaerobic NO2- production was counteracted most by Ca2+ and to a lesser extent by Mg2+; Na+ was without effect. NH4+ at higher concentrations (12 and 15 mM) significantly depressed nitrite production both by roots precultivated in a solution containing besides NH4+ only K+, and by roots precultivated in a full nutrient solution containing K+, Ca2+ and Mg2+, however at lower NH4+ concentrations (0.6 and 2mMNH4+; 15mMNO3-) the decrease was more conspicuous in the KNO3 solution than in the full nutrient solution. Nitrate reductase level was not influenced by this pretreatment. When 6% and 7.5% n-propanol, which increases membrane permeability and causes mixing of storage and metabolic nitrate pools in the cells, was added to the assay medium lacking nitrate, anaerobic nitrite production increased and the differences caused by the precultivation disappeared. These results show that higher K+ concentrations in unbalanced one-salt-solutions of KNO3 can cause higher membrane permeability by accentuating Ca8+ deficiency, which results in a faster penetration of NO3- from the storage pool to the sites of its reduction and in an easier penetration of NO2- out of the roots, and that higher NH4+ concentrations can change nitrate compartmentation and diminish the metabolic NO3- pool which results in a slower nitrate reduction. Besides that, lower NH4+ concentrations in KNO3 solutions (15mMNO3-) probably partially counteract the K+ dependent increase in membrane permeability. The results obtained show that there is no simple, direct relationship between the so-called metabolic pool of nitrate (i.e. anaerobic nitrite production) and the level of nitrate reductase, but that the velocity of nitrate reduction can be influenced by nitrate compartmentation in the cell.     

Intracellular location of nitrate reductase and nitrite reductase. II. Wheat roots

Approximately 15% of the total nitrite reductase of crude homogenates of wheat roots applied to sucrose gradients was separated with an organelle whose isopycnic density was about 1.22 g·cm⁻³. The activity recovered in the supernatant was thought to be particulate in origin, because similar ratios of activity of isoenzyme 1 and 2 of nitrite reductase were found in both particulate and supernatant fractions. The particle with nitrite reductase activity also contained glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triose phosphate isomerase and NADPH diaphorase. This root particle and whole chloroplasts from leaves had a similar isopycnic density as well as these enzymes, and thus the data suggest that the root particle may be a proplastid.

Nitrate reductase was found only in the supernatant and it was not associated with any of the root organelles.

Mitochondria from wheat roots had an equilibrium density of 1.18 g·cm₋₃ and contained both NAD and NADP glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, triosephosphate isomerase and NADPH diaphorase but not nitrite reductase. Microbodies of wheat roots had an equilibrium density of about 1.20 g·cm⁻³ on the sucrose gradient and contained catalase and glycollate oxidase.     

Photosynthesis and the induction of nitrate reductase and nitrite reductase in bean leaves

Summary In laaves of Phaseolus vulgaris L. cv. Prelude, the light-induced increase in activity of NADH-nitrate oxidoreductase (E.C.1.6.6.2; NAR) and reduced benzylviologennitrite oxidoreductase (E.C.1.6.6.4; NIR) starts at a certain stage in the development of the chloroplasts. In leaves with completely developed chloroplasts, a higher increase in activity of NAR and NIR is observed, after induction by the addition of nitrate, in the light than in the dark. DCMU inhibits the increase in activity of the two enzymes in the light. Both in the light in the presence of DCMU, and in the dark the increase in activity reaches a higher level by the addition of sucrose.Induction of NAR, but not of NIR, can be observed in excised etiolated leaves. No induction is found in leaves of intact etiolated seedlings.The relation between photosynthetic reactions and the increase in activity of NAR and NIR is discussed. It is suggested that NADH, indirectly formed by photosynthesis, protects NAR and affects in this way the balance between synthesis and breakdown of the enzyme. The increase in activity of NIR is possibly influenced by the presence of reduced ferredoxin.Abbreviations CAP D-threo-chloramphenicol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - NAR nitrate reductase - NIR nitrite reductase     

Utilization of exogenous sugars by excised maize embryos in culture

Sucrose was markedly superior to fructose and glucose in promoting growth of plantlets from immature maize embryos. The elongation of roots is shown to be more sucrose dependent than that of shoots. On the other hand, the exogenous sucrose was less effective than fructose as substrate for carbohydrate catabolism and for the synthesis of alcohol-insoluble compounds at the beginning of embryo cultivation. The absorbed fructose was found to be rapidly converted to sucrose and the level of endogenous sucrose derived from sugar supplied to the medium was higher in fructosethan in sucrose-fed embryos. The preferential utilization of fructose over sucrose, however, declined with the progress of germination which may be related to the decrease in proportion of scutellum in total mass and physiological activity of the embryo.     

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.     

The effect of some ammonium salts on nitrate reductase level,onin vivo nitrate reduction and on nitrate content in excisedpisum sativum roots

The effect of some ammonium salts on nitrate reductase (NR) level, onin vivo nitrate reduction and on nitrate content was followed in the presence of nitrate in the medium, under changing experimental conditions, in excisedPisum sativum roots, and their effect was compared with that of KNO₃, Ca(NO₃)₂ and NaNO₃ at 15 mM NO₃ ^- concentration, i.e. at a concentration which considerably exceeded the level of saturation with nitrate with respect to nitrate reductase. The effect of ammonium salts on NR level is indirect and changes from a positive one to a strongly negative one which is dependent on the time of action of the salt, on the presence of other cations, on pH of the solution of the ammonium salt and on the nature of the anion of the ammonium salt. A positive effect on the enzyme level can be observed in the presence of other cations than NH₄ ⁺ at suitable concentrations of those ammonium salts, the solutions of which have their pH values in the acid region (i.e. NH₄H₂PO₄, (NH₄)₂SO₄ and NH₄NO₃). However their positive effect is independent of the presence of NH₄ ⁺ ions, and it is obviously the result of an increased concentration of H⁺ ions. A clear-cut negative effect on NR level can be observed after 24 h in one-salt NH₄NO₃ solution where NH₄ ⁺ is not balanced with other cations and thus certainly can adversely influence many metabolic processes, and in the solutions containing neutral (pH 6.2) and dibasic ammonium phosphates in which dissolved undissociated ammonia [(NH₃). (H₂O) which can also affect many metabolic processes incl. proteosynthesis] probably has a toxic influence. Thein vivo nitrate reduction is always depressed in excised pea roots in the presence of ammonium salts in the medium, regardless of the level of nitrate reductase. Under the described conditions, no relationship could be established between the enzyme level and the so-called metabolic NO₃ ^- pool (i.e. NO₂ ^- production under anaerobic conditions), nor between NR level and the total nitrate content in the roots. One-salt solutions of NaNO₃, Ca(NO₃)₂ and KNO₃ exert different effects on the level of nitrate reductase and on the content of NO₃ ^- in the roots, but the in vivo NO₃ ^- reduction shows the same trend as NR level in the roots influenced by these salts. Cl^- ions, supplied in NH₄C1, depress both NR level and NO₃ ^- content in the roots at higher concentrations, but they do not significantly affect the in vivo nitrate reduction in comparison with other ammonium salts. These results indicate that NR level,in vivo nitrate reduction, and nitrate uptake can be regulated in pea roots independently of each other.     

The quiescent centre in excised roots ofPisum sativum L.

Protoplasma - Excised roots ofPisum sativum were cultured in White's medium supplemented with various concentrations of sucrose or IAA and exposed to3H-TdR for 24 hours, three days after...     

The effect of exogenously supplied hydroxylamine on glutamate dehydrogenase,nitrate reductase,and nitrite reductase in isolated pea roots

Hydroxylamine added to the nutrient medium in sublethal concentrations (0.2 to 1.0 mN) enhanced NADH₂ dependent glutamate dehydrogenase activity in isolated pea roots. The increase in activity depended on proteosynthesis and was lower in the presence of NO₃ ^? and NH₄ ⁺ ions. The induction of nitrate reductase and of nitrite reductase was partly inhibited by sublethal hydroxylamine concentrations.     

Effect of chloramphenicol and cycloheximide on the induction of nitrate reductase and nitrite reductase in bean leaves

Summary In etiolated leaves of Phaseolus vulgaris L. cv. Prelude only low levels of NADH-nitrate oxidoreductase (E.C. 1.6.6.2; NAR) and reduced benzyl viologen-nitrite oxidoreductase (E.C. 1.6.6.4; NIR) could be detected, even in the presence of nitrate. When nitrate was available illumination of leaves of 10-day-old etiolated seedlings resulted in an induction of both NAR and NIR. In the absence of nitrate no induction of the enzymes took place, although greening of the leaves was normal. Chloramphenicol (CAP) and cycloheximide (CHI), applied at the beginning of the light period, inhibited the induction of both NAR and NIR. Administered after 24 h of illumination CHI still inhibited the induction of both enzymes whereas CAP was no longer inhibitory. The induction of NAR and NIR by nitrate in green leaves in light was inhibited by CHI but not by CAP. From these results it seems likely that both the enzymes NAR and NIR are synthesized on cytoplasmic ribosomes. Before the enzymes can be manufactured in the cytoplasm some chloroplast development is required.Abbreviations CAP chloramphenicol - CHI cycloheximide - G-6-P(-dh) glucose-6-phosphate (dehydrogenase) - NAR nitrate reductase - NIR nitrite reductase