The possibility of photo-induced induction of nitrate reductase in rice seedlings

Nitrate reductase activity in rice seedlings showed daily fluctuations.Seedlings placed in the dark slowly lost activity and quicklyregained it when exposed to sunlight. Etiolated seedlings alsoshowed rapid increases in activity when transferred to sunlight.This increase in activity by sunlight was inhibited by bothchloramphenicol and ethionine. Ethionine inhibition was reversedby methionine. Purification of nitrate reductase was carriedout by ammonium sulfate fractionation and DEAE-cellulose columnchromatography. Nitrate reductase was purified about 40-foldfrom plants placed in the dark and in sunlight. Incorporationof mediionine-S-¹⁴CH₃ into the nitrate reductase fraction wasstudied. In sunlight, the specific radioactivity of the nitratereductase fraction from the DEAE-cellulose column increased2-fold as compared with that of the crude extracts. Specificradioactivity did not increase in the dark. ¹Present address: Asahi Kasei Chemicals Industry Co., Ltd.,Sameshima 2-1, Fuji, Shizuoka, Japan (Received December 3, 1968; )     

Variation in the nitrate reductase of rice seedlings

Summary Nitrate reductase was induced in rice seedlings by nitrate and by chloramphenicol. During the induction period the different enzyme activities associated with nitrate reductase increased to different degrees. Nitrate induced high NADH-nitrate reductase activity and a great increase in the NADH-cytochrome c reductase activity which was associated with the nitrate reductase in a sucrose gradient. Chloramphenicol induced a nitrate reductase which had higher activity with NADPH than NADH. Chloramphenicol also induced a marked increase in NADPH-cytochrome c reductase activity as well as in NADH-cytochrome c reductase activity. Both activities were associated with the nitrate reductase in a sucrose gradient.After partial purification by sucrose gradient sedimentation or by starch gel electrophoresis, the nitrate reductase of rice induced by nitrate and chloramphenicol showed the same preference in pyridine nucleotide cofactors as was shown by the crude enzyme extracts.     

The induction of nitrate reductase and the preferential assimilation of ammonium in germinating rice seedlings

Nitrate reduotase is induced by nitrate in excised embryos and germinating intact seedlings of rice (Oryza sativa L.). The enzyme is induced 24 hr after imbibition. The rate of enzyme formation increases with the age of seedlings. There is a lag period of 30 to 40 min between the addition of substrate and the formation of nitrate reductase. Formation of the enzyme is promoted by the presence of ammonium. Chloramphenicol, actinomycin D and cycloheximide effectively inhibit the formation of nitrate reductase.     

Effect of carbon dioxide on nitrate accumulation and nitrate reductase induction in corn seedlings

Exposure of the leaf canopy of corn seedlings (Zea mays L.) to atmospheric CO₂ levels ranging from 100 to 800 μl/l decreased nitrate accumulation and nitrate reductase activity. Plants pretreated with CO₂ in the dark and maintained in an atmosphere containing 100 μl/l CO₂ accumulated 7-fold more nitrate and had 2-fold more nitrate reductase activity than plants exposed to 600 μl/l CO₂, after 5 hours of illumination. Induction of nitrate reductase activity in leaves of intact corn seedlings was related to nitrate content. Changes in soluble protein were related to in vitro nitrate reductase activity suggesting that in vitro nitrate reductase activity was a measure of in situ nitrate reduction. In longer experiments, levels of nitrate reductase and accumulation of reduced N supported the concept that less nitrate was being absorbed, translocated, and assimilated when CO₂ was high. Plants exposed to increasing CO₂ levels for 3 to 4 hours in the light had increased concentrations of malate and decreased concentrations of nitrate in the leaf tissue. Malate and nitrate concentrations in the leaf tissue of seven of eight corn genotypes grown under comparable and normal (300 μl/l CO₂) environments, were negatively correlated. Exposure of roots to increasing concentrations of potassium carbonate with or without potassium sulfate caused a progressive increase in malate concentrations in the roots. When these roots were subsequently transferred to a nitrate medium, the accumulation of nitrate was inversely related to the initial malate concentrations. These data suggest that the concentration of malate in the tissue seem to be related to the accumulation of nitrate.     

Effects of univalent cations on the formation of nitrate reductase and nitrite reductase in rice seedlings

An investigation was made to determine the effects of univalentcations as activators on the formation of nitrate reductaseand nitrite reductase in rice seedlings. K⁺ functioned moreeffectively as a univalent cation activator than did other univalentcations examined. Substitution of Rb⁺ for K⁺ resulted in stimulationof nitrate reductase formation at about half the rate obtainedwith K⁺. There was no effect on nitrite reductase formation.Na⁺ could be partially substituted for K⁺ in the formation ofboth enzymes. NH₄⁺ slightly inhibited formation of the enzymes.In the absence of univalent cations, enzyme formation proceededat a slower rate during the initial 15-hr period, but thereafterproceeded at a higher rate. This delayed formation was not observedin the presence of K⁺. Results from inhibitor experiments suggestthat K⁺ stimulates the formation of nitrate reductase and nitritereductase. In conclusion, when nitrate nitrogen is supplied to rice plantsutilization of the nitrogen may be accelerated by increasedformation of enzymes involved in nitrate assimilation in thepresence of K⁺. (Received February 21, 1969; )     

Role of light in the synthesis of nitrate reductase and nitrite reductase in rice seedlings

1. In rice seedlings synthesis of methyl viologen-nitrite reductase was stimulated by light, as was that of NADH-nitrate oxidoreductase (EC 1.6.6.1). A small residual effect of light on the synthesis of the enzymes persisted in the dark for a short time. 2. In etiolated seedlings exposed to light and nitrate, a lag period of 3h was necessary before enzyme synthesis commenced, whereas in green seedlings kept in the dark for 36h, synthesis of both the enzymes started as soon as light and nitrate were provided. 3. Experiments with cycloheximide suggested that fresh protein synthesis in light was necessary for formation of active enzymes. Mere activation by light of inactive enzymes or their precursors, was not involved. 4. In green seedlings synthesis of nitrite reductase was more sensitive to chloramphenicol than that of nitrate reductase. In chloramphenicol-treated etiolated seedlings, however, synthesis of both the enzymes was inhibited to the same extent on subsequent light-treatment. 5. A close correlation was observed between inhibition of the Hill reaction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and simazin [2-chloro-4,6-bis(ethylamino)-s-triazine] (at high concentration) and the inhibition of enzyme synthesis. At lower concentrations, however, simazin stimulated nitrate reductase. 6. In a single leaf synthesis of enzymes was observed only in portions exposed to light, whereas little activity was present in the dark covered part. 7. CO(2) deprivation severely inhibited the synthesis of enzymes in the light. Sucrose could not reverse this effect. 8. In excised embryos cultured in synthetic media containing sucrose, light was also essential for enzyme formation. 9. It is suggested that redox changes taking place in the green tissues as a result of the Hill reaction create conditions favourable for the induced synthesis of nitrate reductase and nitrite reductase.     

Activation of nitrate reductase from rice seedlings by NADH

Rice leaf nitrate reductase was specifically activated by preincubation both at 0° and 25°, with low concentrations of NADH. The nucleotide acted as a positive effector of the enzyme after a time lag of 20 min. NADPH, FMNH₂ and NAD were without any effect.     

Nitrate reductase of rice seedlings and its induction by organic nitro-compounds

Nitrate simultaneously induced NADH- and NADPH-nitrate reductase activities in rice seedlings. Chloramphenicol, other organic nitro-compounds such as o-nitroaniline and 2,4-dinitrophenol and nitrite also induced nitrate reductase in rice seedlings. The nitrate- or nitrite-induced nitrate reductase could accept electrons more efficiently from NADH than NADPH. However, when this enzyme was induced by organic nitro-compounds, it could accept electrons more efficiently from NADPH than NADH.     

Incorporation of nitrate nitrogen in rice seedlings transferred to anaerobic conditions

Summary Incorporation of¹⁵NO₃- into amino acids was studied in 3-day-old aerobic rice seedlings (with coleoptile and root) subjected for 24h to anaerobic conditions. The incorporation of¹⁵N into glutamate, glutamine and alanine accounted for 89% and 84% of total incorporation in coleoptile and root, respectively. These findings indicate that, after the primary incorporation of¹⁵N into glutamate and glutamine, the main fate of nitrate nitrogen in rice seedlings subjected to anoxia is alanine.     

Induction of nitrate reductase in pumpkin seedlings

Nitrate reductase activity (NRA) was found to be induced in9-day-old pumpkin seedlings by nitrate and light. NRA was greatestin leaves and cotyledons and in vitro measurements gave highervalues than in vitro measurements. NRA was found in roots bythe in vivo method but not by the in vitro method. NRA changedwith the age of the seedling with maximum activity in 7-day-oldcotyledons and 9-day-old roots of light grown plants; and rootsof 7-day-old etiolated plants. Little activity was found inetiolated cotyledons. ¹ Present address: College of Agriculture and Animal Husbandry,P.O. Box 32, Rezaiyeh, Iran (Received September 30, 1977; )     

Effect of mycorrhiza and nitrate nutrition on nitrate reductase activity in Scots pine seedlings

In vivo nitrate reductase (EC 1.6.6.1) activity was measured in seedlings of Scots pine ( Pinus sylvestris L.) inoculated with Cenococcum geophilum (Sow.) Ferd. & Winge, Paxillus involutus (Batsch:Fr) Fr, Piloderma croceum Erikss, & Hjortst, and Suillus variegatus (Fr.) O. Kuntze. The activity was higher in the mycorrhizal pine roots than was previously found in the fungus symbiont alone, but lower than in the roots of nonmycorrhizal pine seedlings. The differences observed in a previous study between the fungal species under pure culture conditions were not found in the present work for mycorrhiza synthezised with the same fungal species. An increase in the nitrate concentration of the nutrient solution increased the proportion of the nitrate reductase activity in the needles. The mycorrhizal root tips had higher nitrate reductase activity than nonmycorrhizal root tips in the same root system.     

Distribution of nitrate reductase in ageing bean seedlings

The in vivo activity of nitrate reductase (NR, E.C. 1.6.6.1 [EC] )in the roots, stem and leaves of bean (Phaseolus vulgaris L.)was measured at different ages of seedlings. The leaves alwayshad higher levels of the enzyme than the roots or stem. Thelevel of the enzyme in the very young leaves were low, increasingto a maximum by day 10 to 11 of seedling growth at 26°C,after which it start to decline. The level of the enzyme in7 dayold decotyledonized leaves was about 2.5 times higher thanthat in leaves from intact seedlings. A supply of exogenousnitrate caused a considerable increase in the total organicnitrogen in the leaf only after day 9, when the nitrogen supplyfrom the cotyledons presumably is low. (Received March 22, 1975; )     

Effects of red, far red, and blue light on enhancement of nitrate reductase activity and on nitrate uptake in etiolated rice seedlings

The effects of red (R), far red (FR), or blue light (B) on the enhancement of nitrate reductase (NR) activity and on nitrate uptake in etiolated rice seedlings were examined. On 5-minute illumination followed by 12-hour dark, R caused marked increase of NR activity, but FR and B caused only slight increase. Illumination with 560 ergs per square centimeter per second of R for 5 minutes caused maximal increase. The effect of R was almost completely counteracted by subsequent illumination with 2,000 ergs per square centimeter per second of FR for 10 minutes, indicating that NR induction was mediated by phytochrome. Exogenous supply of inducer nitrate was not required during the 5-minute illumination and the R-FR cycles, if the seedlings were transferred to nitrate solution at the beginning of the dark incubation. NR activity in the shoots was found high when shoots were illuminated but was low when only roots were illuminated. On continuous illumination for 12 hours, B had more effect on NR increase than R.     

Effect of glutamine on the induction of nitrate reductase

Nitrate reductase (NR. EC 1.6.6.1/2) is a substrate inducible enzyme that could be repressed by its end product glutamine or amino acids. To test this hypothesis, 6-day-old maize seedlings ( Zea mays cv. W64A × W182E) were grown hydroponically in a 1/10 strength Hougland's salt solution modified to contain no nitrogen. Previous experiments had established that after a 24-h induction with NO₃⁻ (5 mM KNO₃⁻) the level of NR activity and protein had reached a constant level. In the present experiments when glutamine (5 mM) was included together with NO₃⁻, there was a significant reduction in NR activity (34% of the control values). NR protein and NR mRNA accumulation in the root. In the shoot, on the other hand, glutamine additions had little or no effect on the levels of either NR activity (81% of control) or NR protein. Inhibition of glutamine synthetase by methionine sulfoximine (MSX) resulted in reduced levels of glutamine in both root and shoot tissues. Contrary in our prediction, however, it had no effect on NR activity and mRNA content in roots. In the shoot, on the other hand, there was a marked reduction of NR activity (34% of the control value) and NR protein, but no apparent effect on NR mRNA. When detached shoots were treated with MSX and other inhibitors of glutamine synthetase (tabtoxinine-β-lactam or phosphinothricin) the induction of NR activity by NO₃⁻ was also inhibited. Glutamine additions 15 or 50 mM to detached shoots had essentially no effect on the induction of NR activity (90% of control). These results demonstrate that the influence of glutamine and MSX on the induction of NR in maize root and shoot tissues, respectively, is very different.     

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     

Effect of ammonium and nitrate on growth and appearance of nitrate reductase and nitrite reductase in dark- and light-grown mustard seedlings

Nitrate-induced and phytochrome-modulated appearance of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of the mustard (Sinapis alba L.) seedling is strongly affected by externally supplied ammonium (NH ₄ ⁺ ). In short-term experiments between 60 and 78 h after sowing it was found that in darkness NH ₄ ⁺ —simultaneously given with NO ₃ ^- —strongly inhibits appearance of nitrate-inducible NR and NIR whereas in continuous far-red light—which operates exclusively via phytochrome without significant chlorophyll formation —NH ₄ ⁺ (simultaneously given with NO ₃ ^- ) strongly stimulates appearance of NR. The NIR levels are not affected. This indicates that NR and NIR levels are regulated differently. In the absence of external NO ₃ ^- appearance of NR is induced by NH₄ in darkness as well as in continuous far-red light whereas NIR levels are not affected. On the other hand, in the absence of external NO ₃ ^- , exogenous NH ₄ ⁺ strongly inhibits growth of the mustard seedling in darkness as well as in continuous far-red light. This effect can be abolished by simultaneously supplying NO ₃ ^- . The adverse effect of NH ₄ ⁺ on growth (NH ₄ ⁺ -toxicity) cannot be attributed to pH-changes in the medium since it was shown that neither the growth responses nor the changes of the enzyme levels are related to pH changes in the medium. Non-specific osmotic effects are not involved either.Abbreviations c continuous - D darkness - FR far-red light - NIR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.6.6.1)