Comparative Induction of Nitrate Reductase by Nitrate and Nitrite in Barley Leaves

The comparative induction of nitrate reductase (NR) by ambient NO₃⁻ and NO₂⁻ as a function of influx, reduction (as NR was induced) and accumulation in detached leaves of 8-day-old barley (Hordeum valgare L.) seedlings was determined. The dynamic interaction of NO₃⁻ influx, reduction and accumulation on NR induction was shown. The activity of NR, as it was induced, influenced its further induction by affecting the internal concentration of NO₃⁻. As the ambient concentration of NO₃⁻ increased, the relative influences imposed by influx and reduction on NO₃⁻ accumulation changed with influx becoming a more predominant regulant. Significant levels of NO₃⁻ accumulated in NO₂⁻-fed leaves. When the leaves were supplied cycloheximide or tungstate along with NO₂⁻, about 60% more NO₃⁻ accumulated in the leaves than in the absence of the inhibitors. In NO₃⁻-supplied leaves NR induction was observed at an ambient concentration of as low as 0.02 mm. No NR induction occurred in leaves supplied with NO₂⁻ until the ambient NO₂⁻ concentration was 0.5 mm. In fact, NR induction from NO₂⁻ solutions was not seen until NO₃⁻ was detected in the leaves. The amount of NO₃⁻ accumulating in NO₂⁻-fed leaves induced similar levels of NR as did equivalent amounts of NO₃⁻ accumulating from NO₃⁻-fed leaves. In all cases the internal concentration of NO₃⁻, but not NO₂⁻, was highly correlated with the amount of NR induced. The evidence indicated that NO₃⁻ was a more likely inducer of NR than was NO₂⁻.     

Comparative studies on the induction and inactivation of nitrate reductase in corn roots and leaves

A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH₂-nitrate reductase and NO₃⁻-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO₃⁻ was supplied. WO₄⁼ suppressed the induction of NADH- and FMNH₂-nitrate reductase activities in root tips and leaves. The NO₃⁻-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO₄⁼. In leaves, on the other hand, there was a marked superinduction of the NO₃⁻-induced NADH-cytochrome c reductase in the presence of WO₄⁼.     

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite

An NADPH-dependent NO₂⁻-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP⁺ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO₂⁻ reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO₂⁻ reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP⁺. NADP⁺ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO₂⁻ reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO₂⁻ reduction and (b) consume NADP⁺, releasing FNR from NADP⁺ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO₂⁻ reduction observed in vitro is capable of accounting for the observed rates of dark NO₃⁻ assimilation by C. reinhardtii.     

Effect of Nitrate and Ammonium Nutrition of Nonnodulated Phaseolus vulgaris L. on Phosphoenolpyruvate Carboxylase and Pyruvate Kinase Activity

Young bean plants (Phaseolus vulgaris L. var Saxa) were fed with 3.5 or 10 millimolar N in either the form of NO₃⁻ or NH₄⁺, after being grown on N-free nutrient solution for 8 days. The pH of the nutrient solutions was either 6 or 4. The cell sap pH and the extractable activities of phosphoenolpyruvate carboxylase and of pyruvate kinase from roots and primary leaves were measured over several days.

The extractable activity of phosphoenolpyruvate carboxylase (based on soluble protein) from primary leaves increased with NO₃⁻ nutrition, whereas with NH₄⁺ nutrition and on N-free nutrient solution the activity remained at a low level. Phosphoenopyruvate carboxylase activity from the roots of NH₄⁺-fed plants at pH 4 was finally somewhat higher than from the roots of plants grown on NO₃⁻ at the same pH. There was no difference in activity from the root between the N treatments when pH in the nutrient solutions was 6. The extractable activity of pyruvate kinase from roots and primary leaves seemed not to be influenced by the N nutrition of the plants.

The results are discussed in relation to the physiological function of both enzymes with special regard to the postulated functions of phosphoenolpyruvate carboxylase in C3 plants as an anaplerotic enzyme and as part of a cellular pH stat.

     

The Uptake of NO3?, NO2?, and NH4+ by Intact Wheat (Triticum aestivum) Seedlings : I. Induction and Kinetics of Transport Systems

The inducibility and kinetics of the NO₃⁻, NO₂⁻, and NH₄⁺ transporters in roots of wheat seedlings (Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for and the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO₃⁻ and NO₂⁻ transporters. Evidence for substrate inducibility of the NH₄⁺ transporters requiring 5 hours is presented. The transport of NO₃⁻ was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO₂⁻ and NH₄⁺. The K_m values for NO₃⁻, NO₂⁻, and NH₄⁺ were, respectively, 0.027, 0.054, and 0.05 millimolar. The K_m for mechanism II of NO₃⁻ transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar.     

Nitrate inhibition of nodulation can be overcome by the ethylene inhibitor aminoethoxyvinylglycine

Previously, we reported (a) a positive correlation between the nitrate concentrations in growth medium and ethylene evolved from uninoculated and inoculated alfalfa (Medicago sativa) roots and (b) a negative correlation between ethylene evolution and nodulation. Here, we report that the inhibitory effect of NO₃⁻ on nodulation of alfalfa can be eliminated by the ethylene inhibitor aminoethoxyvinylglycine (AVG). This effect was probably related to the strong inhibition (90%) of ethylene biosynthesis caused by AVG in these inoculated and NO₃⁻-treated roots. These results support our hypothesis that the inhibitory effect of NO₃⁻ is mediated through the phytohormone ethylene. A possible role of endogenous ethylene in the autoregulation of nodulation also is discussed. AVG at 10 micromolar significantly (P < 0.05) increased total nitrogenase activity (acetylene reduction) in 2.5 and 5 millimolar NO₃⁻-fed plants probably as a result of the very high stimulation of nodulation.     

Comparative Kinetics and Reciprocal Inhibition of Nitrate and Nitrite Uptake in Roots of Uninduced and Induced Barley (Hordeum vulgare L.) Seedlings

Nitrate and NO₂⁻ transport by roots of 8-day-old uninduced and induced intact barley (Hordeum vulgare L. var CM 72) seedlings were compared to kinetic patterns, reciprocal inhibition of the transport systems, and the effect of the inhibitor, p-hydroxymercuribenzoate. Net uptake of NO₃⁻ and NO₂⁻ was measured by following the depletion of the ions from the uptake solutions. The roots of uninduced seedlings possessed a low concentration, saturable, low K_m, possibly a constitutive uptake system, and a linear system for both NO₃⁻ and NO₂⁻. The low K_m system followed Michaelis-Menten kinetics and approached saturation between 40 and 100 micromolar, whereas the linear system was detected between 100 and 500 micromolar. In roots of induced seedlings, rates for both NO₃⁻ and NO₂⁻ uptake followed Michaelis-Menten kinetics and approached saturation at about 200 micromolar. In induced roots, two kinetically identifiable transport systems were resolved for each anion. At the lower substrate concentrations, less than 10 micromolar, the apparent low K_ms of NO₃⁻ and NO₂⁻ uptake were 7 and 9 micromolar, respectively, and were similar to those of the low K_m system in uninduced roots. At substrate concentrations between 10 and 200 micromolar, the apparent high K_m values of NO₃⁻ uptake ranged from 34 to 36 micromolar and of NO₂⁻ uptake ranged from 41 to 49 micromolar. A linear system was also found in induced seedlings at concentrations above 500 micromolar. Double reciprocal plots indicated that NO₃⁻ and NO₂⁻ inhibited the uptake of each other competitively in both uninduced and induced seedlings; however, K_i values showed that NO₃⁻ was a more effective inhibitor than NO₂⁻. Nitrate and NO₂⁻ transport by both the low and high K_m systems were greatly inhibited by p-hydroxymercuribenzoate, whereas the linear system was only slightly inhibited.     

Differential light induction of nitrate reductases in greening and photobleached soybean seedlings

Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO₃⁻, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO₃⁻ and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO₃⁻ and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO₃⁻. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO₃⁻, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO₃⁻ requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO₃⁻ requirement for induction were quite different. K_m values for NO₃⁻ were identical for both nitrate reductases.     

Biphasic Behavior of Anammox Regulated by Nitrite and Nitrate in an Estuarine Sediment

The production of N₂ gas via anammox was investigated in sediment slurries at in situ NO₂⁻ concentrations in the presence and absence of NO₃⁻. With single enrichment above 10 μM ¹⁴NO₂⁻ or ¹⁴NO₃⁻ and ¹⁵NH₄⁺, anammox activity was always linear (P < 0.05), in agreement with previous findings. In contrast, anammox exhibited a range of activity below 10 μM NO₂⁻ or NO₃⁻, including an elevated response at lower concentrations. With 100 μM NO₃⁻, no significant transient accumulation of NO₂⁻ could be measured, and the starting concentration of NO₂⁻ could therefore be regulated. With dual enrichment (1 to 20 μM NO₂⁻ plus 100 μM NO₃⁻), there was a pronounced nonlinear response in anammox activity. Maximal activity occurred between 2 and 5 μM NO₂⁻, but the amplitude of this peak varied across the study (November 2003 to June 2004). Anammox accounted for as much as 82% of the NO₂⁻ added at 1 μM in November 2003 but only for 15% in May 2004 and for 26 and 5% of the NO₂⁻ added at 5 μM for these two months, respectively. Decreasing the concentration of NO₃⁻ but holding NO₂⁻ at 5 μM decreased the significance of anammox as a sink for NO₂⁻. The behavior of anammox was explored by use of a simple anammox-denitrification model, and the concept of a biphasic system for anammox in estuarine sediments is proposed. Overall, anammox is likely to be regulated by the availability of NO₃⁻ and NO₂⁻ and the relative size or activity of the anammox population.     

Intercellular localization of nitrate reductase in roots

Experiments were conducted with segments of corn roots to investigate whether nitrate reductase (NR) is compartmentalized in particular groups of cells that collectively form the root symplastic pathway. A microsurgical technique was used to separate cells of the epidermis, of the cortex, and of the stele. The presence of NR was determined using in vitro and enzyme-linked immunosorbent assays. In roots exposed to 0.2 millimolar NO₃⁻ for 20 hours, NR was detected almost exclusively in epidermal cells, even though substantial amounts of NO₃⁻ likely were being transported through cortical and steler cells during transit to the vascular system. Although NR was present in all cell groups of roots exposed to 20.0 millimolar NO₃⁻, the majority of the NR still was contained in epidermal cells. The results are consistent with previous observations indicating that limited reduction of endogenous NO₃⁻ occurs during uptake and reduction of exogenous NO₃⁻. Several mechanisms are advanced to account for the restricted capacity of cortical and stelar cells to induce NR and reduce NO₃⁻. It is postulated that (a) the biochemical system involved in the induction of NR in the cortex and stele is relatively insensitive to the presence of NO₃⁻, (b) the receptor for the NR induction response and the NR protein are associated with cell plasmalemmae and little NO₃⁻ is taken up by cells of the cortex and stele, and/or (c) NO₃⁻ is compartmentalized during transport through the symplasm, which limits exposure for induction of NR and NO₃⁻ reduction.     

Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis

Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO₃⁻ or NH₄⁺, the solutions being maintained at pH 5.5. In NO₃⁻-fed plants excess nutrient anion over cation uptake was equivalent to net OH⁻ efflux, and the total charge from NO₃⁻ and SO₄²⁻ reduction equated to the sum of organic anion accumulation plus net OH⁻ efflux. In NH₄⁺-fed plants a large H⁺ efflux was recorded in close agreement with excess cation over anion uptake. This H⁺ efflux equated to the sum of net cation (NH₄⁺ minus SO₄²⁻) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO₃⁻ that is taken up and reduced in NO₃⁻-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH₄⁺-fed plants absorbed NH₄⁺ was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO₃⁻-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients.     

Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase

Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO₃⁻ medium were characterized. The in vivo NO₃⁻ reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO₂⁻ reductase as compared with the wild type. This coincided with higher accumulation of NO₂⁻ by the variant than by the wild type cells after transfer from alanine medium to NO₃⁻ medium. NO₂⁻ accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions.     

Root respiration associated with nitrate assimilation by cowpea

Nitrate uptake by roots of cowpea (Vigna unguiculata) was measured using ¹⁵NO₃⁻, and the energy cost to the root was estimated by respirometry. Roots of 8-day-old cowpea seedlings respired 0.6 to 0.8 milligram CO₂ per plant per hour for growth and maintenance. Adding 10 millimolar NO₃⁻ to the root medium increased respiration by 20 to 30% during the following 6 hours. This increase was not observed if the shoots were in the dark. Removal of NO₃⁻ from the root medium slowed the increase of root respiration. The ratios of additional respiration to the total nitrogen uptake and reduced nitrogen content in roots were 0.4 gram C per gram N and 2.3 grams C per gram N, respectively. The latter value is close to theoretical estimates of nitrate assimilation, and is similar to estimates of 1 to 4 grams C per gram N for the respiratory cost of symbiotic N₂ fixation.     

Early effects of salinity on nitrate assimilation in barley seedlings

The effect of NaCl and Na₂SO₄ salinity on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var Numar) seedlings was studied. The induction of the NO₃⁻ transporter was affected very little; the major effect of the salts was on its activity. Both Cl⁻ and SO₄²⁻ salts severely inhibited uptake of NO₃⁻. When compared on the basis of osmolality of the uptake solutions, Cl⁻ salts were more inhibitory (15-30%) than SO₄²⁻ salts. At equal concentrations, SO₄²⁻ salts inhibited NO₃⁻ uptake 30 to 40% more than did Cl⁻ salts. The absolute concentrations of each ion seemed more important as inhibitors of NO₃⁻ uptake than did the osmolality of the uptake solutions. Both K⁺ and Na⁺ salts inhibited NO₃⁻ uptake similarly; hence, the process seemed more sensitive to anionic salinity than to cationic salinity.

Unlike NO₃⁻ uptake, NO₃⁻ reduction was not affected by salinity in short-term studies (12 hours). The rate of reduction of endogenous NO₃⁻ in leaves of seedlings grown on NaCl for 8 days decreased only 25%. Nitrate reductase activity in the salt-treated leaves also decreased 20% but its activity, determined either in vitro or by the `anaerobic' in vivo assay, was always greater than the actual in situ rate of NO₃⁻ reduction. When salts were added to the assay medium, the in vitro enzymic activity was severely inhibited; whereas the anaerobic in vivo nitrate reductase activity was affected only slightly. These results indicate that in situ nitrate reductase activity is protected from salt injury. The susceptibility to injury of the NO₃⁻ transporter, rather than that of the NO₃⁻ reduction system, may be a critical factor to plant survival during salt stress.

     

Characteristics of Injury and Recovery of Net NO3? Transport of Barley Seedlings from Treatments of NaCl

The nature of the injury and recovery of nitrate uptake (net uptake) from NaCl stress in young barley (Hordeum vulgare L, var CM 72) seedlings was investigated. Nitrate uptake was inhibited rapidly by NaCl, within 1 minute after exposure to 200 millimolar NaCl. The duration of exposure to saline conditions determined the time of recovery of NO₃⁻ uptake from NaCl stress. Recovery was dependent on the presence of NO₃⁻ and was inhibited by cycloheximide, 6-methylpurine, and cerulenin, respective inhibitors of protein, RNA, and sterol/fatty acid synthesis. These inhibitors also prevented the induction of the NO₃⁻ uptake system in uninduced seedlings. Uninduced seedlings exhibited endogenous NO₃⁻ transport activity that appeared to be constitutive. This constitutive activity was also inhibited by NaCl. Recovery of constitutive NO₃⁻ uptake did not require the presence of NO₃⁻.     

Characterization of Nitrite Degradation by Lactobacillus casei subsp. rhamnosus LCR 6013

Nitrites are potential carcinogens. Therefore, limiting nitrites in food is critically important for food safety. The nitrite degradation capacity of Lactobacillus casei subsp. rhamnosus LCR 6013 was investigated in pickle fermentation. After LCR 6013 fermentation for 120 h at 37°C, the nitrite concentration in the fermentation system was significantly lower than that in the control sample without the LCR 6013 strain. The effects of NaCl and Vc on nitrite degradation by LCR 6013 in the De Man, Rogosa and Sharpe (MRS) medium were also investigated. The highest nitrite degradations, 9.29 mg/L and 9.89 mg/L, were observed when NaCl and Vc concentrations were 0.75% and 0.02%, respectively in the MRS medium, which was significantly higher than the control group (p ≤ 0.01). Electron capture/gas chromatography and indophenol blue staining were used to study the nitrite degradation pathway of LCR 6013. The nitrite degradation products contained N₂O, but no NH₄ ⁺The LCR 6013 strain completely degraded all NaNO₂ (50.00 mg/L) after 16 h of fermentation. The enzyme activity of NiR in the periplasmic space was 2.5 times of that in the cytoplasm. Our results demonstrated that L. casei subsp. rhamnosus LCR 6013 can effectively degrade nitrites in both the pickle fermentation system and in MRS medium by NiR. Nitrites are degraded by the LCR 6013 strain, likely via the nitrate respiration pathway (NO₂ ⁻>NO⁻>N₂O⁻>N₂), rather than the aammonium formation pathway (dissimilatory nitrate reduction to ammonium, DNRA), because the degradation products contain N₂O, but not NH₄ ⁺.     

Effect of Light and NO(3) on Wheat Leaf Phosphoenolpyruvate Carboxylase Activity: Evidence for Covalent Modulation of the C(3) Enzyme

Phosphoenolpyruvate carboxylase (PEPcase) activity was studied in excised leaves of wheat (Triticum aestivum L.) in the dark and in the light, in presence of either N-free (low-NO₃⁻ leaves) or 40 millimolar KNO₃ (high-NO₃⁻ leaves) nutrient solutions. PEPcase activity increased to 2.7-fold higher than that measured in dark-adapted tissue (control) during the first 60 minutes and continued to increase more slowly to 3.8-fold that of the control. This level was reached after 200 minutes exposure of the leaves to light and high NO₃⁻. In contrast, the lower rate of increase recorded for low-NO₃⁻ leaves ceased after 60 minutes of exposure to light at 2.3-fold the control level. The short-term NO₃⁻ effect increased linearly with the level of NO₃⁻ uptake. In immunoprecipitation experiments, the antibody concentration for PEPcase precipitation increased with the protein extracts from the different treatments in the order: control, illuminated low-NO₃⁻ leaves, illuminated high-NO₃⁻ leaves. This order also applied with regard to a decreasing sensitivity to malate and an increasing stimulation by okadaic acid (an inhibitor of P-protein phosphatases). Following these studies, ³²P labeling experiments were carried out in vivo. These showed that the light-induced change in the properties of the PEPcase was due to an alteration in the phosphorylation state of the protein and that this effect was enhanced in high-NO₃⁻ conditions. Based on the responses of PEPcase and sucrose phosphate synthase in wheat leaves to light and NO₃⁻, an interpretation of the role of NO₃⁻ as either an inhibitor of P-protein phosphatase(s) or activator of protein kinase(s) is inferred. In the presence of NO₃⁻, the phosphorylation state of both PEPcase and sucrose phosphate synthase is increased. This causes activation of the former enzyme and inhibition of the latter. We suggest that NO₃⁻ modulates the relative protein kinase/protein phosphatase ratio to favor increased phosphorylation of both enzymes in order to redirect carbon flow away from sucrose synthesis and toward amino acid synthesis.     

Inhibition of Nitrate Transport by Anti-Nitrate Reductase IgG Fragments and the Identification of Plasma Membrane Associated Nitrate Reductase in Roots of Barley Seedlings

Membrane associated nitrate reductase (NR) was detected in plasma membrane (PM) fractions isolated by aqueous two-phase partitioning from barley (Hordeum vulgare L. var CM 72) roots. The PM associated NR was not removed by washing vesicles with 500 millimolar NaCl and 1 millimolar EDTA and represented up to 4% of the total root NR activity. PM associated NR was stimulated up to 20-fold by Triton X-100 whereas soluble NR was only increased 1.7-fold. The latency was a function of the solubilization of NR from the membrane. NR, solubilized from the PM fraction by Triton X-100 was inactivated by antiserum to Chlorella sorokiniana NR. Anti-NR immunoglobulin G fragments purified from the anti-NR serum inhibited NO₃⁻ uptake by more than 90% but had no effect on NO₂⁻ uptake. The inhibitory effect was only partially reversible; uptake recovered to 50% of the control after thorough rinsing of roots. Preimmune serum immunoglobulin G fragments inhibited NO₃⁻ uptake 36% but the effect was completely reversible by rinsing. Intact NR antiserum had no effect on NO₃⁻ uptake. The results present the possibility that NO₃⁻ uptake and NO₃⁻ reduction in the PM of barley roots may be related.     

Soybean mutants lacking constitutive nitrate reductase activity : I. Selection and initial plant characterization

The objectives of this study were to select and initially characterize mutants of soybean (Glycine max L. Merr. cv Williams) with decreased ability to reduce nitrate. Selection involved a chlorate screen of approximately 12,000 seedlings (progeny of mutagenized seed) and subsequent analyses for low nitrate reductase (LNR) activity. Three lines, designated LNR-2, LNR-3, and LNR-4, were selected by this procedure.

In growth chamber studies, the fully expanded first trifoliolate leaf from NO₃⁻-grown LNR-2, LNR-3, and LNR-4 plants had approximately 50% of the wild-type NR activity. Leaves from urea-grown LNR-2, LNR-3, and LNR-4 plants had no NR activity while leaves from comparable wild-type plants had considerable activity; the latter activity does not require the presence of NO₃⁻ in the nutrient solution for induction and on this basis is tentatively considered as a constitutive enzyme. Summation of constitutive (urea-grown wild-type plants) and inducible (NO₃⁻-grown LNR-2, LNR-3, or LNR-4 plants) leaf NR activities approximated activity in leaves of NO₃⁻-grown wild-type plants. Root NR activities were comparable in wild-type and mutant plants grown on NO₃⁻, and roots of both plant types lacked constitutive NR activity when grown on urea. In both growth chamber- and field-grown plants, oxides of nitrogen [NO_(x)] were evolved from young leaves of wild-type plants, but not from leaves of LNR-2 plants, during in vivo NR assays. Analysis of leaves from different canopy locations showed that constitutive NR activity was confined to the youngest three fully expanded leaves of the wild-type plant and, therefore, on a total plant canopy basis, the NR activity of LNR-2 plants was approximately 75% that of wild-type plants. It is concluded that: (a) the NR activity in leaves of NO₃⁻-grown wild-type plants includes both constitutive and inducible activity; (b) the missing NR activity in LNR-2, LNR-3, and LNR-4 leaves is the constitutive component; and (c) the constitutive NR activity is associated with NO_(x) evolution and occurs only in physiologically young leaves.