Soybean Mutants Lacking Constitutive Nitrate Reductase Activity : II. Nitrogen Assimilation, Chlorate Resistance, and Inheritance

Nitrogen assimilation in three nitrate reductase (NR) mutants of soybean (Glycine max L. Merr. cv Williams) was studied in the growth chamber and in the field. These mutants, LNR-2, LNR-3, and LNR-4, lack the non-NO₃⁻-inducible or constitutive fraction of leaf NR activity found in wild-type plants, but this had no effect on the concentration of nitrogen accumulated when grown on NO₃⁻ in the growth chamber. Dry weight accumulation of two of the mutants (LNR-3 and LNR-4) was decreased relative to LNR-2 and wild type. In the field, LNR-2 had dry weights and nitrogen concentrations similar to the wild type at 34 and 61 days after planting, and at maturity. Acetylene reduction activities were also similar at 61 days.     

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.     

Characterization of Nitrate Reductase Deficient Mutants of Chlorella sorokiniana

After x-ray irradiation, 13 mutants of Chlorella sorokiniana incapable of using NO₃⁻ as N source were isolated using a pinpoint method. Using immunoprecipitation and Western blot assays, no nitrate reductase was found in five strains while in eight mutants the enzyme was detected. The latter strains contained different patterns of nitrate reductase partial reactions. All isolates were of the nia-type as indicated by the inducibility of purine hydroxylase I and by complementation of nitrate reductase activity in the Neurospora crassa mutant Nit-1. A restoration of NADP-nitrate reductase in Nit-1 was also obtained with NH₄⁺-grown cells indicating that Mo-cofactor is constitutive in Chlorella. Complementation experiments among the Chlorella mutants resulted in restoration of NADH-nitrate reductase activity. The characteristics of some of the Chlorella mutants are discussed in view of an improper orientation of Mo-cofactor in the residual nitrate reductase protein.     

Purification and Characterization of NAD(P)H:Nitrate Reductase and NADH:Nitrate Reductase from Corn Roots

The nitrate reductase activity of 5-day-old whole corn roots was isolated using phosphate buffer. The relatively stable nitrate reductase extract can be separated into three fractions using affinity chromatography on blue-Sepharose. The first fraction, eluted with NADPH, reduces nearly equal amounts of nitrate with either NADPH or NADH. A subsequent elution with NADH yields a nitrate reductase which is more active with NADH as electron donor. Further elution with salt gives a nitrate reductase fraction which is active with both NADH and NADPH, but is more active with NADH. All three nitrate reductase fractions have pH optima of 7.5 and Stokes radii of about 6.0 nanometers. The NADPH-eluted enzyme has a nitrate K_m of 0.3 millimolar in the presence of NADPH, whereas the NADH-eluted enzyme has a nitrate K_m of 0.07 millimolar in the presence of NADH. The NADPH-eluted fraction appears to be similar to the NAD(P)H:nitrate reductase isolated from corn scutellum and the NADH-eluted fraction is similar to the NADH:nitrate reductases isolated from corn leaf and scutellum. The salt-eluted fraction appears to be a mixture of NAD(P)H: and NADH:nitrate reductases.     

Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons

The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5- to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO₃. Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO₃, subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO₃-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active.     

Regulation of Nitrate Reductase Activity in Leaves of Soybean Bragg and Its Nodulated Mutants

The extracts from leaves of nodulated soybean (Glycine max (L.) Merr. ) cv. Bragg and its nodulated mutants i. e. non-nodulated Nod 49, supernodulated nts 382 and nts 246 contained inhibitors of activities iNR, c1NR and c2NR in vitro. Both white light illumination of 300 μE · m-2 · s-1 and inoculation with strain USDAll0 were essential conditions for ac- cumulating these inhibitors in leaves. Comparing inhibiting activities of the extracts from different varieties indicated that Nod 49 extract showed stronger inhibition than Bragg extract did, but nts 382 extract had only weakest inhibitory effect. The inoculated Bragg root extract possessed the same inhibitory activity as its leaf extract. The inoculated nts 382 root extract, like its leaf extract, showed only a little inhibitory activity. However inoculated Nod 49 root extract lead to an inhibition of leaf c2NR activity, which was different from its leaf extract that inhibited three kinds of NR activities. The above results suggested that both leaf and root extracts contained common inhibitory factor which was accumulated after inoculation.     

Characteristics of a Nitrate Reductase in a Barley Mutant Deficient in NADH Nitrate Reductase

A barley (Hordeum vulgare L.) mutant, nar1a (formerly Az12), deficient in NADH nitrate reductase activity is, nevertheless, capable of growth with nitrate as the sole nitrogen source. In an attempt to identify the mechanism(s) of nitrate reduction in the mutant, nitrate reductase from nar1a was characterized to determine whether the residual activity is due to a leaky mutation or to the presence of a second nitrate reductase. The results obtained indicate that the nitrate reductase in nar1a differs from the wild-type enzyme in several important aspects. The pH optima for both the NADH and the NADPH nitrate reductase activities from nar1a were approximately pH 7.7, which is slightly greater than the pH 7.5 optimum for the NADH activity and considerably greater than the pH 6.0 to 6.5 optimum for the NADPH activity of the wild-type enzyme. The nitrate reductase from nar1a exhibits greater NADPH than NADH activity and has apparent K_m values for nitrate and NADH that are approximately 10 times greater than those of the wild-type enzyme. The nar1a nitrate reductase has apparent K_m values of 170 micromolar for NADPH and 110 micromolar for NADH. NADPH, but not NADH, inhibited the enzyme at concentrations greater than 50 micromolar.     

Nitrate Reductase Complex of Escherichia coli K-12: Isolation and Characterization of Mutants Unable to Reduce Nitrate

Thirty-eight mutants unable to reduce nitrate were isolated from Escherichia coli and characterized biochemically and genetically. All of the mutants exhibited reduced or insignificant levels of formate dehydrogenase, nitrate reductase, or various combinations of these activities and cytochrome b(1) under conditions which resulted in the production of high levels of these activities by the wild-type parental strains. Most of the mutants reverted readily to wild type, and all mapped within a restricted region on the chromosome linked to the tryptophan genes. It was proposed that nitrate reduction in E. coli was catalyzed exclusively by an organized complex containing formate dehydrogenase, cytochrome b(1), and nitrate reductase.     

Purification and Kinetics of Higher Plant NADH:Nitrate Reductase

Squash cotyledon (Cucurbita pepo L.) NADH:nitrate reductase (NR) was purified 150-fold with 50% recovery by a single step procedure based on the affinity of the NR for blue-Sepharose. Blue-Sepharose, which is prepared by direct coupling of Cibacron blue to Sepharose, appears to bind squash NR at the NADH site. The NR can be purified in 2 to 3 hours to a specific activity of 2 μmol of NADH oxidized/minute • milligram of protein. Corn (Zea mays L.) leaf NR was also purified to a specific activity of 6.9 μmol of NADH oxidized/minute • milligram of protein using a blue-Sepharose affinity step. The blue-Sepharose method offers the advantages of a rapid purification of plant NR to a high specific activity with reasonable recovery of total activity.

The kinetic mechanism of higher plant NR was investigated using these highly purified squash and corn NR preparations. Based on initial velocity and product inhibition studies utilizing both enzymes, a two-site ping-pong mechanism is proposed for NR. This kinetic mechanism incorporates the concept of the reduced NR transferring electrons from the NADH site to a physically separated nitrate site.

     

Nitrate Reductase mRNA Regulation in Nicotiana plumbaginifolia Nitrate Reductase-Deficient Mutants

Light and substrate regulation of nitrate reductase (NR) expression were compared in wild type and mutant lines of Nicotiana plumbaginifolia. Mutants affected in the NR structural gene (nia) or in the biosynthesis of the NR molybdenum cofactor (cnx) were examined. nia mutants expressing a defective apoenzyme, as well as cnx mutants, overexpressed NR mRNA, whereas nia mutants devoid of detectable NR protein had reduced or undetectable NR mRNA levels. Diurnal fluctuations of NR mRNA were specifically abolished in nia and cnx mutants, suggesting that the integrity of NR catalytic activity is required for the expression of diurnal oscillations. Unlike some fungal mutants, the nia and cnx mutants examined retained nitrate inducibility of NR expression. The possibility of autogenous control of NR expression in higher plants is discussed.     

Isolation and Initial Characterization of Constitutive Nitrate Reductase-Deficient Mutants NR328 and NR345 of Soybean (Glycine max)

Two nitrate reductase deficient mutants of soybean (Glycine max [L.] Merr. cv Bragg) were isolated from approximately 10,000 M₂ seedlings, using a direct enzymic assay in microtiter plates. Stable inheritance of NR345 and NR328 phenotypes has been demonstrated through to the M₅ generation. Both mutants were affected in constitutive nitrate reductase activity. Assayable activities of cNR in nitrate-free grown seedlings was about 3 to 4% of the control for NR345 and 14 to 16% of the control for NR328. Both mutants expressed inducible NR during early plant development and were sensitive to nitrate and urea inhibition of nodulation. These new mutants will allow an extension of the characterization of nitrate reductases and their function in soybean. Preliminary evidence indicates that NR345 is similar to the previously isolated mutant nr₁, while NR328 is different.     

Developmental and Biochemical Regulation of 'Constitutive' Nitrate Reductase Activity in Leaves of Nodulating Soybean

The developmental profile of ‘constitutive’ nitratereductase activity (cNRA) in leaves of soybean (Glycine max(L.) cv. Bragg) plants at different ages is described. The youngestleaves had most cNRA and the activity dropped off as a newerleaf developed above it. Each leaf had its distinct active periodof in vivo cNRA. This pattern was different in urea-grown andsymbiotically-grown plants (inoculated with Bradyrhizobium japonicumstrain USDA 110), where the latter had no detectable in vivocNRA in older leaves. Urea-grown plants maintained considerablein vivo NRA in such older leaves. When symbiotically-grown plantshad their nodules removed, in vivo cNRA reappeared in olderleaves within 1 d of removal, nearly reaching levels of youngleaves at 3 d after nodule excision. Allantoic acid (ALL), oneof the known transport ureides of soybeans, was implicated asa possible signal molecule from nodules to leaves. Allantoicacid (100 µM) inhibited in vitro c₁ NRA significantly,with 400 µM ALL resulting in complete inhibition. In contrast,allantoin (ALN) had no inhibitive effect on NRA. Inhibitionof c₁NRA by ALL was by a competitive process, judging from Lineweaver-Burkeplots against nitrate. Kinetics showed a constant Vmax of around105 nmol NO₂ mg^–1 protein h^–1 and a K_m for nitrateof 15 mM, which increased to 60 mM in the presence of 200 µMallantoic acid. Non-specific (ionic and pH-related) influenceswere eliminated. Allantoic acid also had a slight stimulatingeffect of in vitro NRA (up about 25% at 400 µM). Thesefindings suggest that c1NRA may be involved in ureide metabolism,rather than in vivo nitrate metabolism. Key words: Root-shoot interaction, nitrogen metabolism, nodulation, symbiosis     

Sunflower Nitrate Reductase: Purification and Characterization

A single isoform, NADH: nitrate reductase (NR), was purified 500 folds from sunflower leaves by affinity chromatography on Blue Sepharose CL-6B. Purified NR had a pH optima of 7.25 and a molecular weight of 210 kD. In SDS-PAGE, two bands of 47 and 56 kD were obtained. NADH: ferric citrate reductase activity was copurified with NR with a specific activity of 2. The Vmax of NADH: ferric citrate reductase was 8.69 units mg^-1 protein and the apparent Km for ferric citrate was 0.435 mM.     

Genetic and Biochemical Analysis of Intragenic Complementation Events among Nitrate Reductase Apoenzyme-Deficient Mutants of Nicotiana Plumbaginifolia

Intragenic complementation has been observed between apoenzyme nitrate reductase-deficient mutants (nia) of Nicotiana plumbaginifolia. In vivo as in vitro, the NADH-nitrate reductase (NR) activity in plants heterozygous for two different nia alleles was lower than in the wild type plant, but the plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of homozygous nia mutants was restored by mixing extracts from two complementing nia mutants. These observations suggest that NR intragenic complementation results from either the formation of heteromeric NR or from the interaction between two modified enzymes. Complementation was only observed between mutants retaining different partial catalytic activities of the enzyme. Results are in agreement with molecular data suggesting the presence of three catalytic domains in the subunit of the enzyme.     

Purification of Squash NADH:Nitrate Reductase by Zinc Chelate Affinity Chromatography

NADH:nitrate reductase (EC 1.6.6.1) was isolated and purified from the green cotyledons of 5-day-old squash seedlings (Cucurbita maxima L.). The 10-hour purification procedure consisted of two steps: direct application of crude enzyme to blue Sepharose and specific elution with NADH followed by direct application of this effluent to a Zn²⁺ column with elution by decreasing the pH of the phosphate buffer from 7.0 to 6.2. The high specific activity (100 micromoles per minute per milligram protein) and high recovery (15-25%) of electrophoretically homogeneous nitrate reductase show that the enzyme was not damaged by exposure to the bound zinc. With this procedure, homogeneous nitrate reductase can be obtained in yields of 0.5 milligram per kilogram cotyledons.     

Inactivation of Sunflower NADH:Nitrate Reductase by White Light-Activated Rose Bengal

Chemical modification of purified nitrate reductase (NR) from sunflower leaves by white light-irradiated rose bengal was studied. NADH:NR activity was inhibited by light-activated rose bengal in both a concentration- and time-dependent manner. MV:NR activity was less sensitive to inhibition than NADH:NR activity, especially when the enzyme was preincubated with NADH. Preincubation of the enzyme with FAD protected inhibition of NADH:NR activity but not the MV:NR activity. These results suggest that sunflower NR contains sensitive histidine residue which interacts with reduced FAD during catalytic electron transfer. Most importantly, NADH-reduced NR was more sensitive to the irradiated dye, indicating that conformation of the oxidized and reduced enzyme forms were different.     

Magnesium and Calcium Inhibition of Squash Leaf NADH Nitrate Reductase

This paper describes the first experimental evidence that theinhibition of nitrate reductase by Mg²⁺ or Ca²⁺ is related tothe hysteretic properties of the enzyme. The low activity formof nitrate reductase, i.e. the form of nitrate reductase showinghysteretic behaviour, was inhibited 70–90% by 5 mM Ca²⁺or Mg²⁺. However, no inhibition by Ca²⁺ or Mg²⁺ was seen afterthe enzyme was converted to its high activity form by preincubationwith substrates. Addition of thiol compounds or certain aminoacids to the assay mixture also prevented the Mg²⁺ or Ca²⁺ inhibition. (Received June 28, 1993; Accepted August 11, 1993)     

Primary Analysis of Components in Different Soybean Nodulating Mutants and their Effects on Nitrate Reductase Activity and Nodulation

The water extracts of leaves and roots from supernodulating soybean (Glycine max (L.) Merr. ) nts 382 and nonnodulating soybean Nod 49 have been chromatographed using filtering method through the column (25 cm × 2 cm) Sephadex G25 and 4 fractions, namly, nts 382 (Nod 49) F1, nts 382 (Nod 49) F2, nts 382 (Nod 49) F3, and nts 382 (Nod 49) F4 could be distinguished according to nitrate reductase (NR) activities inhibited by the eluate. The inhibition of NR activity by the noninoculated nts 382 F2 and the nts 382 F4 in vitro were much stronger than that by the inoculated nts 382 F2 and nts 382 F4. On the contrary, the obvious inhibition of NR activity in vitro by the noninoculated Nod 49 F2 and Nod 49 F4 were substantialy strengthed again by the innoculated Nod 49 F2 and Nod 49 F4. The facts indicated that the quantity of NR inhibitors in the leaf cells of soybean nts 382 reduced after the inoculation but was that in the inoculated Nod 49 leaf cells further more accumulated. Both nodulations assays, the nodulation of soybean "Bragg " injected with inoculated nts 382 Fl, nts 382 F2, nts 382 F3 and nts 382 F4 from leaves and roots and the nodulation of soybean nts 382 injected with inoculated Nod 49 F2, Nod 49 F3 and Nod 49 F4 from leaves only showed that nts 382 Fl and nts 382 F2 increased nodules of soybean "Bragg" by 1 to 3 times but nts 382 F3 and nts 382 F4 did not. Inhibition of soybeannts 382 nodulation by inoculated Nod 49 F2 Nod 49 F3 and Nod 49 F4 expressed that the Nod 49 F4 only inhibited the nodulation strongly by one time in the experiments with nts 382 plants with leaves, and by 15 times in the experiments with nts 382 plants without leaves at 10 d of inoculation and injection and this inhibition was nonreversible even after stopping injection from the 11th day to the 15th day after inoculation.     

Nitrate Reductase from the Marine Diatom Skeletonema costatum (Biochemical and Immunological Characterization)

Assimilatory nitrate reductase (NR) was purified from the marine diatom Skeletonema costatum (clone Skel) using Cibacron blue-Sepharose affinity chromatography. The single-step purification scheme yielded a 103-fold purification of specific activity with an overall recovery of 40.8%. Only NADH-dependent NR activity (form EC 1.6.6.1) was observed in this species. Kinetic analysis revealed that this form had apparent Michaelis constants of 3.6 [mu]M for NADH and 295 [mu]M for NO3- when purified from cells grown in NO3--enriched seawater. The S. costatum NR exhibits a pH optimum of 7.4, a temperature optimum of 14[deg]C, and enzyme activity not sensitive to Mg2+ inhibition. The strong temperature dependence of NR activity in S. costatum may contribute to the seasonal and latitudinal distributions and abundances of this bloom-forming species. Chromatographically isolated NR was further purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single polypeptide with an apparent molecular mass of 110 kD. The 110-kD polypeptide was used to generate polyclonal antibodies. The antiserum recognized a single 110-kD polypeptide in western blots of total proteins from S. costatum, as well as the native enzyme. Western blot analysis also revealed an antigenic similarity of NR from two additional diatom species, whereas no cross-reactivity was observed with NR from other phytoplankton taxa, including prymnesiophytes, dinoflagellate, cyanobacterium, and green alga. This result suggests a structural diversity of NR in phytoplankton and identifies the potential for development of taxon-specific NR antisera for ecological studies.