Allelochemical effects on net nitrate uptake and plasma membrane H+-ATPase activity in maize seedlings

Seven-day-old maize seedlings grown in a nitrogen-free hydroponic culture were exposed for 48 h to 0, 100 and 300 μM trans-cinnamic, p-coumaric, ferulic, caffeic acids, umbelliferone and 200 μM KNO₃. Net nitrate uptake was affected by trans-cinnamic, ferulic and p-coumaric acids in a concentration-dependent manner, and trans-cinnamic acid appeared to be the strongest inhibitor. Conversely, at low concentrations, caffeic acid stimulated net nitrate uptake while umbelliferone did not influence it. After 24 h of treatment, plasma membrane H⁺-ATPase activity significantly decreased in a concentration-dependent manner in response to trans-cinnamic, ferulic and p-coumaric acids, while umbelliferone and caffeic acid had no effect on H⁺-ATPase activity.     

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, ¹³N as NO₃ ^-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of ¹³N internally was ammonium and amino acids; the amino acid labeling pattern indicated that ¹³NO₃ ^- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO₃ ^- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K _mvalues, 7 M. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.     

Amino Acid interactions in the regulation of nitrate reductase induction in cotton root tips

Glycine, asparagine, and glutamine inhibited the induction by nitrate of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.). This inhibition was partially or entirely prevented when the inhibitor was applied in combination with any of several other amino acids. Studies of ¹⁴C-labeled amino acid uptake showed that, in most cases, the apparent antagonism resulted simply from competition for uptake. However, certain antagonists did not curtail uptake. The most effective of these were leucine (against all three inhibitors), and isoleucine and valine (against asparagine or glutamine, but not glycine). These results show that interactions among amino acids in the regulation of nitrate reductase induction result from at least two mechanisms, one acting on uptake of inhibitory amino acids, and the other involving true antagonism.     

Effects of Ascorbate on the Oxidation of Derivatives of Hydroxycinnamic Acid and the Mechanism of Oxidation of Sinapic Acid by Cell Wall-Bound Peroxidases

A cell wall fraction isolated from epicotyls of Vigna angularis,which contained both ionically and covalently bound peroxidases,rapidly oxidized p-coumaric, caffeic and ferulic acids and slowlyoxidized sinapic acid. The oxidation of sinapic acid was greatlyenhanced in the presence of p-coumaric, caffeic or ferulic acid.Ascorbate (20 µM) inhibited the oxidation of ferulic acidby about 70% and completely inhibited the oxidation of p-coumaricand ferulic acids. The cell wall fraction was capable of bindingferulic and sinapic acids but not caffeic acid. p-Coumaric acidbound only slightly to cell walls. The oxidation of p-coumaricand ferulic acids by KCl-washed cell walls was inhibited byabout 60% and 10%, respectively, by 20 µM ascorbate, butthe oxidation of caffeic acid was completely inhibited by ascorbateat less than 20 µM. The oxidation of derivatives of hydroxycinnamicacid by peroxidases released from cell walls by washing with1 M KCl was completely inhibited by ascorbate. These resultssuggest that the inhibition by ascorbate depends on the substituentgroup of the phenyl ring of the derivatives of hydroxycinnamicacid when the oxidation reaction is catalyzed by cell wall-boundperoxidases and that the oxidation of sinapic acid is mediatedby phenoxyl radicals of derivatives of hydroxycinnamic acidother than sinapic acid. (Received December 2, 1993; Accepted March 3, 1994)     

Evidence for the involvement of plasma-membrane-bound nitrate reductase in signal transduction during blue-light stimulation of nitrate uptake in Chlorella saccharophila

Nitrate uptake in Chlorella saccharophila (Krüger) Nadson was found to be stimulated by blue light, leading to a doubling of the rate. In the presence of background red light (300 mol photons · m^-2 · s^-1), only 15–20 mol photons · m^-2 · s^-1 of blue light was sufficient to saturate this increased uptake rate. Incubation of Chlorella cells with anti-nitrate-reductase immunoglobulin-G fragments inhibited blue-light stimulation. However, ferricyanide (10 M) doubled and dithiothreitol (100 M) inhibited the stimulatory effect of blue light. Among the protein-kinase inhibitors used, only staurosporine (10 M) prevented the blue-light stimulation. Phosphatase inhibitors were without effect and sodium vanadate totally inhibited nitrate uptake, pointing to an involvement of the plasma-membrane ATPase. Preincubation of the cells with calmodulin antagonists or calcium ionophores did not significantly reduce blue-light stimulation of nitrate uptake. The data are discussed with regard to transduction of the signal for blue-light stimulation of nitrate uptake and the possibility that the plasma-membrane-bound nitrate reductase is the blue-light receptor.Abbreviations Chl chlorophyll - DMSO dimethylsulfoxide - 1,2-DHG 1,2-dihexanoylglycerol - ML-9 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine - NR nitrate reductase - H-7 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine - IgG immunoglobulin G - PFD photon flux density - PM plasma membrane - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide This work was supported by a grant from the Deutsche Forschungs-gemeinschaft to R.T.     

Daily changes in nitrate uptake and metabolism in Capsicum annuum

The diurnal pattern of nitrate uptake by Capsicum annuum L. cv. California Wonder in a constant environment is described by a Fourier harmonic, with the maximum uptake in the middle of the photoperiod and the minimum in the middle of the dark period. Comparison of the uptake pattern with that of nitrate reductase (EC 1.6.6.1.) activity suggests against a direct control of one process by the other. This was confirmed by the observation that the pattern of nitrate reductase activity was not altered by restricting nitrate uptake to one hour per day. Translocation of ¹⁵N from the roots is much greater in the lightperiod than in the dark period. Reduction of ¹⁵N in the leaves occurs in the lightperiod but very little is reduced in the dark period. Amino acid levels showed marked daily fluctuations but in the roots neither amino acids, sucrose, fructose, glucose nor malate showed fluctuations. The amino acid composition of roots and leaves differed: glutamine+glutamate were relatively more important in leaves than in roots whereas alanine was a more important constituent of roots than of leaves.Abbreviation NR nitrate 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     

Utilization of nitrate byBeggiatoa alba

Beggiatoa alba B18LD utilizes both nitrate and nitrite as sole nitrogen sources, although nitrite was toxic above 1 mM.B. alba coupledin vivo acetate oxidation, but not sulfide oxidation, with nitrate and nitrite reduction.B. alba could not, however, grow anaerobically with nitrate as the sole electron acceptor. Furthermore, the incorporation of acetate into macromolecules under anaerobic conditions with nitrate as the sole electron acceptor was less 10% of the incorporation with oxygen as the electron acceptor. The product of nitrate reduction byB. alba was ammonia; N₂ or N₂O were not produced. The nitrate reductase activity inB. alba was soluble and it utilized reduced flavins or methyl viologen and dithionite as electron donors. Pyrimidine nucleotides were not used as in vitro electron donors, either alone or with flavins in coupled assays. TheB. alba nitrate reductase activity was competitively inhibited with chlorate and was only mildly inhibited by azide and cyanide. Nitrate was not required for induction of theB. alba nitrate reductase, and neither oxygen nor ammonia repressed its activity. Thus,B. alba nitrate reductase appears to be an assimilatory nitrate reductase with unusual regulatory properties.Non-standard abbreviations MV Methyl viologen - DT dithionite - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase - PPO 2-diphenyloxazole - POPOP 1,4-(bis)-[2-(5-phenyloxazolyl)] benzene - TCA trichloroacetic acid - CCCP carbonylcyanidem-chlorophenylhydrazone - FCCP carbonylcyanidep-trifluoromethoxyphenylhydrazone - TTFA thenoyltrifluoroacetone - PHEN 1,10-phenanthroline - HOQNO 2-heptyl 4-hydroxyquinoline-n-oxide - 8HQ 8-hydroxyquinoline     

Inhibition of nitrate and nitrite reduction by 2,4-dinitrophenol in ankistrodesmus

Summary As Kessler (1955, 1959) has shown, nitrite reduction by the green alga, Ankistrodesmus braunii is completely inhibited by 10^-3 m 2,4-dinitrophenol. However, although nitrite accumulates in the medium when cultures are supplied with nitrate and dinitrophenol, the reduction of nitrate is not completely insensitive to the inhibitor.Direct measurements show that 2,4-dinitrophenol inhibits nitrate disappearance from the medium by 65–80%. The degree of inhibition increases when the initial nitrate concentration is decreased.It is suggested that inhibition of nitrate assimilation by dinitrophenol is due to inhibition of an active uptake of nitrate by the cells, and that at high nitrate concentrations, a dinitrophenol-insensitive uptake process increases in importance.     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

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.     

Interaction between electron transport at the plasma membrane and nitrate uptake by maize (Zea mays L.) roots

Summary In the present study nitrate uptake by maize (Zea mays L.) roots was investigated in the presence or absence of ferricyanide (hexacyanoferrate III) or dicumarol. Nitrate uptake caused an alkalization of the medium. Nitrate uptake of intact maize seedlings was inhibited by ferricyanide while the effect of dicumarol was not very pronounced. Nitrite was not detected in the incubation medium, neither with dicumarol-treated nor with control plants after application of 100 M nitrate to the incubation solution. In a second set of experiments interactions between nitrate and ferricyanide were investigated in vivo and in vitro. Nitrate (1 or 3 mM) did neither influence ferricyanide reductase activity of intact maize roots nor NADH-ferricyanide oxidoreductase activity of isolated plasma membranes. Nitrate reductase activity of plasma-membrane-enriched fractions was slightly stimulated by 25 M dicumarol but was not altered by 100 M dicumarol, while NADH-ferricyanide oxidoreductase activity was inhibited in the presence of dicumarol. These data suggest that plasma-membrane-bound standard-ferricyanide reductase and nitrate reductase activities of maize roots may be different. A possible regulation of nitrate uptake by plasmalemma redox activity, as proposed by other groups, is discussed.Abbreviations ADH alcohol dehydrogenase - HCF III hexacyanoferrate III (ferricyanide) - ME NADP-dependent malic enzyme - NR nitrate reductase - PM plasma membrane - PM NR nitrate reductase copurifying with plasma membranes     

Regulation of nitrate uptake by amino acids in maize cell suspension culture and intact roots

The effect of amino acids on nitrate transport was studied in Zea mays cell suspension cultures and in Zea mays excised roots. The inclusion of aspartic acid, arginine, glutamine and glycine (15mM total amino acids) in a complete cell-culture media containing 1.0 mM NO₃ ^- strongly inhibited nitrate uptake and the induction of accelerated uptake rates. The nitrate uptake rate increased sharply once solution amino acid levels fell below detection limits. Glutamine alone inhibited induction in the cell suspension culture. Maize seedlings germinated and grown for 7 days in a 15 mM mixture of amino acids also had lower nitrate uptake rates than seedlings grown in 0.5 mM Ca(NO₃)₂ or 1 mM CaCl₂. As amino acids are the end product of nitrate assimilation, the results suggest an end-product feed-back mechanism for the regulation of nitrate uptake.     

Comparative effects of selenite and selenate on nitrate assimilation in barley seedlings

Abstract. The effect of SeO₃ and SeO₄ on NO₃ assimilation in 8-d-old barley (Hordeum vulgare L.) seedlings was studied over a 24-h period. Selenite at 0.1 mol. m^? in the uptake solutions severely inhibited the induction of NO₃ uptake and active nitrate reductases. Selenate, at 1.0 mol m^?3 in the nutrient solution, had little effect on induction of activities of these systems until after 12 h; however, when the seedlings were pretreated with 1.0 mol m^?3 SeO₄ for 24 h, subsequent NO₃ uptake from SeO₄-free solutions was inhibited about 60%. Sulphate partially alleviated the inhibitory effect of SeO₃ when supplied together in the ambient solutions, but had no effect in seedlings pretreated with SeO₃. By contrast, SO₄ partially alleviated the inhibitory effect of SeO₄ even in seedlings pretreated with SeO₄. Since uptake of NO₃ by intact seedlings was also inhibited by SeO₃, the percentage of the absorbed NO₃ that was reduced was not affected. By contrast, SeO₄, which affected NO₃ uptake much less, inhibited the percentage reduced of that absorbed. However, when supplied to detached leaves, both SeO₃ and SeO₄ inhibited the in vivo reduction of NO₃ as well as the induction of nitrate reductase and nitrite reductase activities. Selenite was more inhibitory than SeO₄; approximately a five to 10 times higher concentration of SeO₄ than SeO₃ was required to achieve similar inhibition. In detached leaves, the inhibitory effect of both SeO₃ and SeO₄ on in vivo NO₃ reduction as well as on the induction of nitrate reductase activity was partially alleviated by SO₄. The inhibitory effects of Se salts on the induction of nitrite reductase were, however, completely alleviated by SO₄. The results show that in barley seedlings SeO₃ is more toxic than SeO₄. The reduction of SeO₄ to SeO₃ may be a rate limiting step in causing Se toxicity.     

Inhibition of nitrate uptake by ammonia in a blue-green alga,Anabaena cylindrica

Ammonia at concentrations above 1×10^-5 M inhibits uptake of nitrate in the nitrogen-fixing blue-green alga, Anabaena cylindrica. This inhibition takes place both in the light and in the dark. The rate of nitrate uptake is stimulated by light. Addition of relatively high concentrations of nitrate (1–10 mM) reversibly inhibits ammonia uptake. FCCP, an uncoupler of phosphorylation, inhibits both nitrate and ammonia uptake. Ammonia may inhibit nitrate uptake by reducing the supply of energy (ATP) for active nitrate transport.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxy-phenylhydrazone - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

Rationalization of properties of nitrate reductases in Rhodopseudomonas capsulata

1. The properties of nitrate reductase activities have been compared in several strains of Rhodopseudomonas capsulata grown phototrophically in the presence of nitrate as sole nitrogen source.
2. Strains AD2 and BK5 resemble the spontaneous mutant N22DNAR⁺ (described by McEwan et al. 1982 FEBS Lett. 150, 277\2-280) in that reduction of nitrate was inhibited by either illumination or oxygen but not by NH ₄ ⁺ , and that electron flow to nitrate under dark anaerobic conditions generated a cytoplasmic membrane potential (as judged by an electrochromic shift in the absorbance spectrum of endogenous carotenoid pigments). In contrast disappearance of nitrate from suspensions of strains N22 and St. Louis was dependent upon illumination and was inhibited by NH ₄ ⁺ . Membrane potentials were not generated by addition of nitrate in the dark to N22, St. Louis or strain Kbl.
3. Nitrate reductase was shown to be located in the periplasmic space of both strain AD2 and mutant N22DNAR⁺. The nitrate reductase activity in cells of AD2 and N22DNAR⁺ was relatively insensitive to azide, with 0.5mM azide required for 50% inhibition. The nitrate reductase of strain BK5 was more strongly associated with the cytoplasmic membrane and no conclusion could be reached about whether it was located on the periplasmic or cytoplasmic surface. In BK5 cells nitrate reductase activity was sensitive to low concentrations of azide (50% inhibition with 2 \gmM azide). It is proposed that functionally the nitrate reductase activity in strains AD2, BK5 and N22DNAR⁺ has identical roles. These roles are suggested to include:
4. The first step in the assimilation of nitrate.
5. Provision of an alternative electron acceptor to oxygen for generating a membrane potential.
6. A mechanism for disposing of excess reducing equivalents in the maintenance of balanced growth. This type of nitrate reductase, especially in AD2 and N22DNAR⁺, appears to resemble that described in a denitrifying strain of Rps. sphaeroides, but to differ markedly from its membrane-bound counterpart in other bacteria including the denitrifying Paracoccus denitrificans and Escherichia coli.
7. In other strains of Rps. capsulata including St. Louis, N22 and Kbl, only an assimilatory nitrate reductase, whose activity in intact cells is relatively sensitive to azide, is present in anaerobic, phototrophic cultures grown with nitrate as nitrogen source. As this reductase cannot be detected after breakage of cells, no conclusion can be made as to its location in the cell.

     

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

Pyridine nucleotide specificity and other properties of purified nitrate reductase from Chlorella variegata

Nitrate reductase (NR) (EC 1.6.6.2) from Chlorella variegata 211/10d has been purified by blue sepharose affinity chromatography. The enzyme can utilise NADH or NADPH for nitrate reduction with apparent K _m values of 11.5 M and 14.5 M, respectively. Apparent K _m values for nitrate are 0.13 mM (NADH-NR) and 0.14 mM (NADPH-NR). The diaphorase activity of the enzyme is inhibited strongly by parachloromercuribenzoic acid; NADH or NADPH protects the enzyme against this inhibition. NR proper activity of the enzyme is partially inactive after extraction and may be activated after the addition of ferricyanide. The addition of NAD(P)H and cyanide causes a reversible inactivation of the NR proper activity although preincubation with either NADH or NADH and ADP has no significant effect.Abbreviations NR Nitrate reductase - FAD Flavin-adenine dinucleotide - FMN Riboflavin 5-phosphate - p-CMB para-Chloromercuribenzoic - BV Benzyl viologen     

Genetic control of nitrate reduction in the cyanobacterium nostoc muscorum

Summary There were three kinds of chlorate resistant (Clr-R) mutants: Clr-R₁ type did not show nitrate reductase activity, Clr-R₂ type was deficient in nitrate uptake activity and Clr-R₃ type was defective in both. It is suggested that the genetic determinant of the uptake system is distinct from that of the reductase system.The NH ₄ ⁺ -repressible uptake system showed a requirement of nitrate as an activator of its activity. In comparison, NH ₄ ⁺ -repressible reductase enzyme showed requirement of nitrate neither as an inducer of its synthesis nor as an activator of its activity. Thus, the available mutational and physiological evidence suggest no involvement of the nitrate uptake system in control of nitrate reductase activity and vice versa.Abbreviations Tricine (N-tris[Hydroxymethyl] methyl glycine) - Tris Tris (Hydroxymethyl) amino methane - OD Optical Density - Chl. Chlorophyll