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     

Ferredoxin and ferredoxin-NADP-oxidoreductase in leaves ofPhaseolus vulgaris L.

During light-induced greening of 10-dayold etiolated bean seedlings a strong increase is observed of ferredoxin (Fd) and of ferredoxin-NADP-oxidoreductase (FNR; E.C. 1.6.99.4) activity, both known to reside in chloroplasts. The production of Fd starts immediately upon illumination and proceeds almost linearly for at least the next 72 h. It is inhibited by chloramphenicol (CAP) but not by cycloheximide (CHI), beit that in the presence of the latter Fd synthesis was impaired after 48 h of illumination. We conclude that for the elaboration of functional Fd in greening chloroplasts protein synthesis on chloroplast ribosomes is required. The increase of FNR activity shows a lag of about 24 h and is blocked by both CAP and CHI. When CAP is applied at 24 h after the illumination started it has no effect. We suggest that the synthesis of FNR on cytoplasmic ribosomes requires prior synthesis of protein(s) on chloroplast ribosomes.The nature of possible interactions between chloroplasts and cytoplasm is discussed.Abbreviations CAP D-threo-chloramphenicol - CHI cycloheximide - DCIP dichlorophenol-indophenol - DEAE diethylaminoethyl - Fd Ferredoxin - FNR ferredoxin-NADP-oxidoreductase - NAR nitrate reductase - NIR nitrite reductase     

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.     

The light-induced development of nitrate reductase in etiolated barley shoots: An inhibitory effect of laevulinic acid

Induction of nitrate reductase EC 1.6.6.1 in etiolated barley (Hordeum vulgare L., var. Proctor) required continuous illumination and showed a lag period of about three hours. During the first 16 h of illumination the ratio NADH/NAD and NADPH/NADP, taken as a measure of internal oxidation reduction potential, declined. The inhibitor DCMU applied to whole leaves at concentrations shown to inhibit the reduction of cytochrome f by Photosystem 2 light did not inhibit the induction of nitrate reductase nor did it diminish the ratio of reduced to oxidised puridine nucleotides in the early hours of greening. It was concluded that light driven electron flow was not necessary for nitrate reductase induction. Chloramphenicol gave a slight inhibition of nitrate reductase induction. Laevulinic acid was added to greening barley leaves to inhibit tetrapyrrole pigment biosynthesis and plastid development. It strongly inhibited chlorophyll synthesis and nitrate reductase induction, with relatively little effect upon Photosystem 1 and 2 activities in isolated plastids. The activities of other inducible enzymes and control enzymes were little affected by laevulinic acid. Laevulinic acid also inhibited nitrate reductase induction by added nitrate in fully-greened illuminated plants grown in nitrate-free medium and so is unlikely to be acting through inhibition of plastid development. This inhibitor lowered the level of protohaem in whole leaves and plastids of greening barley and it is postulated that it may diminish the protohaem available for the assembly of a cytochrome b component of nitrate reductase.Abbreviations DCMU 3-(3:4-Dichlorophenyl)-1:1-dimethylurea - LA laevulinic acid     

Inheritance of nitrite reductase and regulation of nitrate reductase, nitrite reductase, and glutamine synthetase isozymes

Banding patterns of nitrate reductase (NR), nitrite reductase (NiR), and glutamine synthetase (GS) from leaves of diploid barley (Hordeum vulgare), tetraploid wheat (Triticum durum), hexaploid wheat (Triticum aestivum), and tetraploid wild oats (Avena barbata) were compared following starch gel electrophoresis. Two NR isozymes, which appeared to be under different regulatory control, were observed in each of the three species. The activity of the more slowly migrating nitrate reductase isozyme (NR1) was induced by NO3- in green seedlings and cycloheximide inhibited induction. However, the activity of the faster NR isozyme (NR2) was unaffected by addition of KNO3, and it was not affected by treatments of cycloheximide or chloramphenicol. Only a single isozyme of nitrite reductase was detected in surveys of three tetraploid and 18 hexaploid wheat, and 48 barley accessions; however, three isozymes associated with different ecotypes were detected in the wild oats. Inheritance patterns showed that two of the wild oat isozymes were governed by a single Mendelian locus with two codominant alleles; however, no variation was detected for the third isozyme. Treatment of excised barely and wild oat seedlings with cycloheximide and chloramphenicol showed that induction of NiR activity was greatly inhibited by cycloheximide, but only slightly by chloramphenicol. Only a single GS isozyme was detected in extracts of green leaves of wheat, barley, and wild oat seedlings. No electrophoretic variation was observed within or among any of these three species. Thus, this enzyme appears to be the most structurally conserved of the three enzymes.     

Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves

The effects of red and far-red light on the enhancement of in vitro nitrate reductase activity and on nitrate accumulation in etiolated excised maize leaves were examined. Illumination for 5 min with red light followed by a 4-h dark period caused a marked increase in nitrate reductase activity, whereas a 5-min illumination with far-red light had no effect on the enzyme activity. The effect of red light was completely reversed by a subsequent illumination with the same period of far-red light. Continuous far-red light also enhanced nitrate reductase activity. Both photoreversibility by red and far-red light and the operation of high intensity reaction under continuous far-red light indicated that the induction of nitrate reductase was mediated by phytochrome. Though nitrate accumulation was slightly enhanced by red and continuous far-red light treatments by 17% and 26% respectively, this is unlikely to account for the entire increase of nitrate reductase activity. The far-red light treatments given in water, to leaves preincubated in nitrate, enhanced nitrate reductase activity considerably over the dark control. The presence of a lag phase and inhibition of increase in enzyme activity under continuous far-red light-by tungstate and inhibitors of RNA synthesis and protein synthesis-rules out the possibility of activation of nitrate reductase and suggests de novo synthesis of the enzyme affected by phytochrome.     

Induction of nitrate reductase by chloramphenicol in detached cucumber cotyledons

Summary Chloramphenicol (CAP) induced nitrate reductase activity (NRA) in detached, etiolated cucumber (Cucumis sativus L.) cotyledons. The effect was reduced by cycloheximide. Light was not necessary for induction of the enzyme but potentiated the effect of CAP as an inducer of NRA, despite the fact that the antibiotic inhibited chlorophyll accumulation.CAP at suboptimal concentrations (2.5–5 mM) acted synergistically with succinic acid-2,2-dimethylhydrazide (B-Nine) and benzylaminopurine (BAP) in respect to induction of NRA, the effect being especially marked in darkness. The highest NRA was found in cotyledons treated for 24 h with a mixture of CAP+BAP+B-Nine. The effects of KNO₃ and CAP on NRA were neither synergistic nor additive.KNO₃ and BAP induced NADH-NR (EC 1.6.6.1). The same was true for CAP and B-Nine, but the latter two compounds induced, in addition, some activity of another NR that could utilize NADPH as an electron donor.     

Nitrate and nitrate reductase in Erythrina caffra seeds: Enhancement of induction by anoxia and possible role in germination

The nitrate level in seed embryonic axes of Erythrina caffra Thunb. which is capable of anaerobic germination, was about 2.5 times higher than in seed axes of Pisum sativum L. a species incapable of anaerobic germination. Nitrate levels in E. caffra seeds decreased during germination and this was not due to leaching. Both NADH- and NADPH-dependent nitrate reductase (NAR) activities increased during germination. The increase was prevented by cycloheximide. The activity of NADH-NAR (EC 1.6.6.1) was higher than that of NADPH-NAR (EC 1.6.6.3). Both NAR activities were higher in anoxia than in air during germination. The NAR activities in Pisum seeds were very much lower than in Erythrina seeds. Anoxia (N₂ or argon) enhanced the induction of NAR by KNO₃ in germinated E. caffra axes. The NADH- and NADPH-NAR activities were induced to equally high levels by KNO₃ under anoxia. The enhancement was depressed by cycloheximide. It is concluded that nitrate and NAR activity may play a role in the anaerobic germination of E. caffra seeds.Abbreviation NAR nitrate reductase Financial support was obtained from the University of the Orange Free State and the Foundation for Research Development     

Nitrate reductase in agrostemma githago comparison of the inductive effects of nitrate and cytokinin

The effect of nitrate and cytokinin on the induction of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in embryos of Agrostemma githago was compared. Increased enzyme levels in response to treatment with the cytokinin benzyladenine were not correlated with a general stimulation of protein synthesis or a general reduction of protein breakdown. Actinomycin D did not inhibit the formation of nitrate reductase in response to nitrate or the cytokinin. Cycloheximide and puromycin inhibited the induction by the hormone to the same extent as, or even more than, the incorporation of [¹⁴C]leucine into protein. Induction of nitrate reductase by nitrate was much less susceptible to inhibition by cycloheximide and puromycin than induction of the enzyme by benzyladenine. When induction of nitrate reductase was carried out in the presence of ²H₂O, isopycnic equilibrium centrifugation in CsCl showed that incorporation of ²H into the enzyme had occured. The increase in the buoyant density of nitrate reductase was the same whether the enzyme was induced by nitrate or by benzyladenine, indicating that at least part of the nitrate reductase molecule was newly synthesized in both instances.     

Light and Nitrate Requirements for Induction of Nitrate Reductase Activity in Hordeum vulgare

The importance of light to the induction of nitrate reductase activity in barley (Hordeum vulgare L.) was studied. Activity in etiolated leaves in darkness stayed at a low endogenous level even while large amounts of nitrate were actively accumulated. Light was required for any increase in activity, though the requirement may be satisfied to a limited extent before nitrate is available. Nitrate reductase activity was induced in the dark in green leaves which had not previously had nitrate but were supplied nitrate at the beginning of the dark period. If the nitrate then made available was sufficient, nitrate reductase activity increased until the effect of the previous light treatment was exhausted. Activity then decreased even though nitrate uptake continued. Upon returning the leaves to light, enzymatic activity increased again, as expected. Nitrate uptake was eliminated as an experimental variable by giving dark-grown plants nitrate, then detaching the leaves for induction studies. Under these conditions light saturation occurred between 3600 and 7700 lux at exemplary periods of illumination. At intensities of 3600 lux and above, activity increased sharply after a 6-hour lag period. As light intensity was decreased below 3600 lux the lag period became longer. Thus, when sufficient nitrate was available, the extent of induction of nitrate reductase activity was regulated by light.     

Rapid activation by phytochrome of nitrate reductase in the cotyledons of Sinapis alba

Summary Nitrate reductase in the cotyledons of etiolated seedlings of Sinapis alba L. responds rapidly to the addition of nitrate. The response is inhibited by cycloheximide at low concentrations. The enzyme is also under phytochrome control. Five minutes of red light irradiation leads instantaneously to a 45% increase in enzyme activity. Increases in activity, linear with respect to time and with no lag phases are promoted by continuous far-red or blue irradiation. These increases are insensitive to cycloheximide. Thus, light and nitrate act through different mechanisms in controlling nitrate reductase activity and phytochrome does not act via controlling the rate of synthesis of the enzyme.Abbreviation cot pr pair of cotyledons     

The interaction of respiration and photosynthesis in induction of nitrate reductase activity

The respiration and photosynthesis requirement for induction and maintenance of nitrate reductase activity was determined on leaves of Hordeum vulgare L. In this induction, glucose substituted for light in both dark-grown and carbohydrate-depleted green leaves. Oxygen appeared to be required for induction in all cases studied. In light and under N₂, 3-(3,4-dichlorophenyl)-1,1-dimethylurea completely inhibited induction, presumably by inhibiting the production of O₂, Hence, under N₂ the leaves appeared to utilize both the O₂ produced by photosynthesis and the CO₂ produced by respiration. CO₂ fixation can then produce both photosynthate to drive the induction and terminal electron acceptors to allow photosynthetic electron flow. This possibility was further suggested by the observation that CO₂ was an absolute requirement for induction in carbohydrate-depleted barley leaves. Results obtained with respiratory inhibitors also indicated that respiration drove the induction of nitrate reductase.     

Phytochrome, nitrate movement, and induction of nitrate reductase in etiolated pea terminal buds

The role of phytochrome in the induction of nitrate reductase of etiolated field peas (Pisum arvense L.) was examined. Terminal bud nitrate concentration increased in darkness, and the increase correlated with induction of nitrate reductase following brief exposure of intact plants to red, blue, far red, and white lights. Brief light exposure of intact plants stimulated nitrate uptake and induction of nitrate reductase by terminal buds subsequently excised and incubated on nitrate solution in darkness; exposure of excised buds in contact with nitrate led to less uptake but more induction. Nitrate and nitrate reductase activity both declined during incubation with water, irrespective of light treatment. Nitrate enrichment of intact terminal buds and uptake into excised buds and increases in nitrate reductase activity were all red/far red reversible. Dimethyl sulfoxide (1%, v/v) and sugars (sucrose 0.5%, glucose 1, w/v), although stimulating nitrate uptake into excised tissue in darkness, failed to enhance nitrate reductase activity over dark controls. Phytochrome may regulate nitrate reductase via both nitrate movement and a general mechanism such as enhancement of protein synthesis.     

Synthesis and degradation of barley nitrate reductase

Nitrate and light are known to modulate barley (Hordeum vulgare L.) nitrate reductase activity. The objective of this investigation was to determine whether barley nitrate reductase is regulated by enzyme synthesis and degradation or by an activation-inactivation mechanism. Barley seedling nitrate reductase protein (cross-reacting material) was determined by rocket immunoelectrophoresis and a qualitative immunochemical technique (western blot) during the induction and decay of nitrate reductase activity. Nitrate reductase cross-reacting material was not detected in root or shoot extracts from seedlings grown without nitrate. Low levels of nitrate reductase activity and cross-reacting material were observed in leaf extracts from plants grown on nitrate in the dark. Upon nitrate induction or transfer of nitrate-grown etiolated plants to the light, increases in nitrate reductase activity were positively correlated with increases in immunological cross-reactivity. Root and shoot nitrate reductase activity and cross-reacting material decreased when nitrate-induced seedlings were transferred to a nitrate-free nutrient solution or from light to darkness. These results indicate that barley nitrate reductase levels are regulated by de novo synthesis and protein degradation.     

Phytochrome regulation of nitrate reductase in wheat

In excised wheat leaves, the activity of nitrate reductase was enhanced by a brief pulse of red light and this increase was reversed by far-red light irradiation. Even under continuous far-red light, nitrate reductase activity increased by 258% after 18 h. When leaves were kept in distilled water during exposure to red light and then transferred to potassium nitrate, there was no difference in endogenous nitrate concentration. The nitrate reductase activity was the same whether leaves were floated in potassium nitrate or in distilled water during irradiation. Partial to complete inhibition of enzyme activity was observed when leaves were incubated in actinomycin-D and cycloheximide respectively, following 4 h of red light irradiation.In vitro irradiation of extract had no significant effect on nitrate reductase activity     

Light-induced Development of Polyribosomes and the Induction of Nitrate Reductase in Corn Leaves

Nitrate reductase activity was induced by nitrate in green corn (Zea mays) leaves in either light or darkness. The induction process required oxygen in darkness but not in light. A light treatment was required before the enzyme could be induced in etiolated leaves.     

Gametogenesis in Chlamydomonas II. Effect of cycloheximide on the induction of sexuality

An inhibitor of protein synthesis, CHI, was used to study therelation of protein synthesis to the induction of sexualityin Chlamydomonas. (1) Freshly prepared CAP and DSm did not inhibitinduction, but CAP solution which had been illuminated causedstrong inhibition. (2) CHI inhibited the induction of sexualityremarkably, but not completely. The induction of sexuality maybe of two types, CHI-sensitive and CHI-insensitive. (3) Vigorousagitation of gametogenic cultures resulted in a marked inhibitionof induction in the presence of CHI, and less in its absence.(4) The amounts of light required for CHI-sensitive and CHI-insensitiveinduction differed; respectively, 5 min of illumination at ca.8,000 lux and 3 hr of illumination at the same intensity. (5)For complete blockage of CHI-sensitive induction, the additionof CHI 50 min before illumination was needed. (6) The activitiesof CHI-sensitive and CHIinsensitive induction changed with theage of the culture. (7) It was supposed that some material(s)inhibiting gametogenesis was present in the medium of the agedculture. Based on these results, a hypothetical scheme for gametogenesisis presented. (Received March 7, 1973; )