Uptake of Nitrate by Mutants of Arabidopsis thaliana, Disturbed in Uptake or Reduction of Nitrate

A study of nitrate and chlorate uptake by Arabidopsis thaliana was made with a wildtype and two mutant types, both mutants having been selected by resistance to high chlorate concentrations. All plants were grown on a nutrient solution with nitrate and/or ammonium as the nitrogen source. Uptake was determined from depletion in the ambient solution. Nitrate and chlorate were able to induce their own uptake mechanisms. Plants grown on ammonium nitrate showed a higher subsequent uptake rate of nitrate and chlorate than plants grown on ammonium alone. Mutant B25, which has no nitrate reductase activity, showed higher rates of nitrate and chlorate uptake than the wildtype, when both types were grown on ammonium nitrate. Therefore, the uptake of nitrate is not dependent on the presence of nitrate reductase. Nitrate has a stimulating effect on nitrate and chlorate uptake, whereas some product of nitrate and ammonium assimilation inhibits uptake of both ions by negative feedback. Mutant B 1, which was supposed to have a low chlorate uptake rate, also has disturbed uptake characteristics for nitrate.     

Somatic Hybridization of Nitrate Reductase Deficient Mutants of Nicotiana tabacum by Protoplast Fusion

Protoplasts were isolated from two mutant cell lines of Nicotiana tabacum L. cv. Gatersleben and fused with the aid of polyethylene glycol. Both mutants lacked nitrate reductase and were thus auxotrophic for reduced nitrogen. The fusion resulted in a high frequency of hybrid cells which were detected by their regained ability to grow in media containing nitrate as sole nitrogen source. Thus, the two mutants were found to complement each other in the hybrids. In control experiments, back mutation and cross-feeding were excluded as possible explanations for the occurrence of cell lines utilizing nitrate. A total of 1061 hybrid lines capable of sustained proliferation were isolated. Some of them were further characterized with respect to nitrate reductase activity, chlorate sensitivity, chromosome number, and shoot formation. The results demonstrate that protoplast fusion can be used for the genetic analysis of cell variants of higher plants and that nitrate reductase-deficient mutants provide efficient selective systems for hybrid cells.     

Evidence for a Second Nitrate Reductase Activity That Is Distinct from the Respiratory Enzyme in Salmonella typhimurium

Significant nitrate reductase activity was detected in mutants of Salmonella typhimurium which mapped at or near chlC and which were incapable of growth with nitrate as electron acceptor. The same mutants were sensitive to chlorate and performed sufficient nitrate reduction to permit anaerobic growth with nitrate as the sole nitrogen source in media containing glucose. The mutant nitrate-reducing protein did not migrate with the wild-type nitrate reductase in polyacrylamide electrophoretic gels. Studies of the electrophoretic mobility in gels of different polyacrylamide concentration revealed that the wild-type and mutant nitrate reductases differed significantly in both size and charge. The second enzyme also differed from the wild-type major enzyme in its response to repression by low pH and its lack of response to repression by glucose. The same mutants were found to be derepressed for nitrite reductase and for a cytochrome with a maximal reduced absorbance at 555 nm at 25°C. This cytochrome was not detected in preparations of the wild type grown under the same conditions. Extracts of these mutants contained normal amounts of the b-type cytochromes which, in the wild type, were associated with nitrate reductase and formate dehydrogenase, respectively, although they could not mediate the oxidation of these cytochromes with nitrate. They were capable of oxidizing the derepressed 555-nm peak cytochrome with nitrate. It is suggested that these mutants synthesize a nitrate-reducing enzyme which is distinct from the chlC gene product and which is repressed in the wild type during anaerobic growth with nitrate.     

Nitrate Reductase and Chlorate Toxicity in Chlorella vulgaris Beijerinck

A study of the growth-inhibiting effect of chlorate on the Berlin strain of Chlorella vulgaris Beijerinck provided complete confirmation of the theory of chlorate toxicity first proposed by Åberg in 1947. Chlorate was toxic to the cells growing on nitrate, and relatively nontoxic to the cells growing on ammonium. The latter cells contained only 0.01 as much NADH-nitrate reductase as the nitrate-grown cells. Chlorate could substitute for nitrate as a substrate of the purified nitrate reductase with Km = 1.2 mm, and V_max = 0.9V_max for nitrate. Bromate, and to a much smaller extent, iodate, also served as alternate substrates. Nitrate is a reversible competitive inhibitor of chlorate reduction, which accounts for the partial reversal, by high nitrate concentrations, of the observed inhibition of cell growth by chlorate. During the reduction of chlorate by NADH in the presence of purified nitrate reductase, there was a progressive, irreversible inhibition of the enzyme activity, presumably brought about by the reduction product, chlorite. Both the NADH-nitrate reductase activity and the associated NADH-cytochrome c reductase activity were inactivated to the same extent by added chlorite. The spectral properties of the cytochrome b₅₅₇ associated with the purified enzyme were not affected by chlorite. The inactivation of the nitrate reductase by chlorite could account for the toxicity of chlorate to cells grown on nitrate, though the destruction of other cell components by chlorite or its decomposition products cannot be excluded.     

Nitrate reductase-deficient mutants in barley : immunoelectrophoretic characterization

Nitrate reductase-deficient barley (Hordeum vulgare L.) mutants were assayed for the presence of a functional molybdenum cofactor determined from the activity of the molybdoenzyme, xanthine dehydrogenase, and for nitrate reductase-associated activities. Rocket immunoelectrophoresis was used to detect nitrate reductase cross-reacting material in the mutants. The cross-reacting material levels of the mutants ranged from 8 to 136% of the wild type and were correlated with their nitrate reductase-associated activities, except for nar 1c, which lacked all associated nitrate reductase activities but had 38% of the wild-type cross-reacting material. The cross-reacting material of two nar 1 mutants, as well as nar 2a, Xno 18, Xno 19, and Xno 29, exhibited rocket immunoprecipitates that were similar to the wild-type enzyme indicating structural homology between the mutant and wild-type nitrate reductase proteins. The cross-reacting materials of the seven remaining nar 1 alleles formed rockets only in the presence of purified wild-type nitrate reductase, suggesting structural modifications of the mutant cross-reacting materials. All nar 1 alleles and Xno 29 had xanthine dehydrogenase activity indicating the presence of functional molybdenum cofactors. These results suggest that nar 1 is the structural gene for nitrate reductase. Mutants nar 2a, Xno 18, and Xno 19 lacked xanthine dehydrogenase activity and are considered to be molybdenum cofactor deficient mutants. Cross-reacting material was not detected in uninduced wild-type or mutant extracts, suggesting that nitrate reductase is synthesized de novo in response to nitrate.     

Nitrate reduction in Aerobacter aerogenes

Summary Mutants of A. aerogenes blocked in aerobic and anaerobic nitrate assimilation and deficient in the reduction of nitrate and chlorate were found to give a positive methylred reaction and no gas formation from glucose. Resting cells, grown anaerobically in minimal medium with glucose, did not show gas production from formate. These results show that these mutants are also deficient in formate hydrogenylase. Revertants could readily be obtained by plating on minimal medium with nitrate as sole nitrogen source, indicating that in these mutants a pleiotropic point mutation is present, which affects both nitrate reductase and formate hydrogeny lase. It is suggested, that these mutants are deficient in the formation of an enzyme complex or particle on which these enzymes are present.     

Contribution of Nitrate Assimilation to the Fitness of Pseudomonas syringae pv. syringae B728a on Plants

The ability of Pseudomonas syringae pv. syringae to use nitrate as a nitrogen source in culture and on leaves was assessed. Substantial amounts of leaf surface nitrate were detected directly and by use of a bioreporter of nitrate on bean plants grown with a variety of nitrogen sources. While a nitrate reductase mutant, P. syringae ΔnasB, exhibited greatly reduced growth in culture with nitrate as the sole nitrogen source, it exhibited population sizes similar to those of the wild-type strain on leaves. However, the growth of the ΔnasB mutant was much less than that of the wild-type strain when cultured in bean leaf washings supplemented with glucose, suggesting that P. syringae experiences primarily carbon-limited and only secondarily nitrogen-limited growth on bean leaves. Only a small proportion of the cells of a green fluorescent protein (GFP)-based P. syringae nitrate reductase bioreporter, LK2(pOTNas4), exhibited fluorescence on leaves. This suggests that only a subset of cells experience high nitrate levels or that nitrate assimilation is repressed by the presence of ammonium or other nitrogenous compounds in many leaf locations. While only a subpopulation of P. syringae consumes nitrate at a given time on the leaves, the ability of those cells to consume this resource would be strongly beneficial to those cells, especially in environments in which nitrate is the most abundant form of nitrogen.     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.     

Nitrate reduction mutants of fusarium moniliforme (gibberella fujikuroi)

Twelve strains of Fusarium moniliforme were examined for their ability to sector spontaneously on toxic chlorate medium. All strains sectored frequently; 91% of over 1200 colonies examined formed chlorate-resistant, mutant sectors. Most of these mutants had lesions in the nitrate reduction pathway and were unable to utilize nitrate (nit mutants). nit mutations occurred in seven loci: a structural gene for nitrate reductase (nit1), a regulatory gene specific for the nitrate reduction pathway (nit3), and five genes controlling the production of a molybdenum-containing cofactor that is necessary for nitrate reductase activity (nit2, nit4, nit5, nit6, nit7). No mutations affecting nitrite reductase or a major nitrogen regulatory locus were found among over 1000 nit mutants. Mutations of nit1 were recovered most frequently (39-66%, depending on the strain) followed by nit3 mutations (23-42%). The frequency of isolation of each mutant type could be altered, however, by changing the source of nitrogen in the chlorate medium. We concluded that genetic control of nitrate reduction in F. moniliforme is similar to that in Aspergillus and Neurospora, but that the overall regulation of nitrogen metabolism may be different.     

Control of Nitrogenase mRNA Levels by Products of Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120

Nitrate inhibited nitrogenase synthesis and heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Inhibition of dinitrogen fixation by nitrate did not take place, however, in nitrate reductase-deficient derivatives of this strain. Hybridization of total RNA isolated from cells grown on different nitrogen sources with an internal fragment of the nifD gene showed that regulation of nitrogenase activity by nitrate is exerted through a negative control of the nitrogenase mRNA levels.     

Nitrate Reductase in Barley Roots under Sterile, Low Oxygen Conditions

Levels of nitrate reductase activity (EC 1.9.6.1.) as high as 11 μmoles nitrite produced/hour gram fresh weight were found in barley (Hordeum vulgare cv. Compana) roots grown under low oxygen conditions. Roots of plants given identical treatment under sterile conditions did not develop the high levels of nitrate reductase activity. The results suggest that the buildup of particulate, reduced viologen-utilizing nitrate reductase reported in barley roots may be caused by bacterial contamination. The nitrate reductase activity in roots grown under low oxygen conditions was not specific for reduced nicotinamide adenine dinucleotide like the assimilatory nitrate reductase (EC 1.6.6.1.) normally found in aerated plant roots.     

Nitrate Nonutilizing Mutants and Vegetative Compatibility Groups in Fusarium poae

Nitrate nonutilizing (nit) mutants were recovered from 24 isolates of Fusarium poae and used to force heterokaryons between these isolates and to determine vegetative compatibility. Between 30 and 90% of the mycelial blocks, cultured on medium containing chlorate, produced nit mutants. The amount of chlorate in the medium altered the frequency and spectrum of nit mutants recovered. Most of the mutants (63%) had lesions at a nitrate reductase structural locus (nit1). Another 30% were mutants at one or more loci that control the production of a molybdenum-containing cofactor necessary for nitrate reductase activity (NitM). A few (6%) of the mutations occurred in a regulatory gene specific for the nitrate reduction pathway (nit3). Pairings between nit1 and NitM mutants were made on minimal medium containing nitrate as the sole nitrogen source. A mutant grows thinly unless it forms a complementary heterokaryon upon contact with another mutant. Heterokaryon formation was indicated by dense growth where the two mutant colonies touched. The 24 isolates could be divided into 13 nonoverlapping vegetative compatibility groups, suggesting that asexual exchange of genetic information within F. poae is subject to significant limitations.     

Stress-induced rearrangement of Fusarium retrotransposon sequences

Rearrangement of Fusarium oxysporum retro- transposon skippy was induced by growth in the presence of potassium chlorate. Three fungal strains, one sensitive to chlorate (Co60) and two resistant to chlorate and deficient for nitrate reductase (Co65 and Co94), were studied by Southern analysis of their genomic DNA. Polymorphism was detected in their hybridization banding pattern, relative to the wild type grown in the absence of chlorate, using various enzymes with or without restriction sites within the retrotransposon. Results were consistent with the assumption that three different events had occurred in strain Co60: genomic amplification of skippy yielding tandem arrays of the element, generation of new skippy sequences, and deletion of skippy sequences. Amplification of Co60 genomic DNA using the polymerase chain reaction and divergent primers derived from the retrotransposon generated a new band, corresponding to one long terminal repeat plus flanking sequences, that was not present in the wild-type strain. Molecular analysis of nitrate reductase-deficient mutants showed that generation and deletion of skippy sequences, but not genomic amplification in tandem repeats, had occurred in their genomes.     

Evidence for multiple nitrate uptake systems in the yeast Hansenula polymorpha

Hansenula polymorpha mutants disrupted in the high-affinity nitrate transporter gene (YNT1) are still able to grow in nitrate. To detect the nitrate transporter(s) responsible for this growth a strain containing disruption of the nitrate assimilation gene cluster and expressing nitrate reductase gene (YNR1) under the control of H. polymorpha MOX1 (methanol oxidase) promoter was used (FM31 strain). In this strain nitrate taken up is transformed into nitrite by nitrate reductase and excreted to the medium where it is easily detected. Nitrate uptake which is neither induced by nitrate nor repressed by reduced nitrogen sources was detected in the FM31 strain. Likewise, nitrate uptake detected in the strain FM31 is independent of both Ynt1p and Yna1p and is not affected by ammonium, glutamine or chlorate. The inhibition of nitrite extrusion by extracellular nitrite suggests that the nitrate uptake system shown in the FM31 strain could also be involved in nitrite uptake.     

The isolation and survival characterization of radiation and chemical-mutagen sensitive mutants of Pseudomonas aeruginosa

Chlorate-resistant mutants of Arabidopsis thaliana were isolated in order to find nitrate reductase-less mutants. It appeared that chlorate resistance in higher plants can arise by mutations concerning two different mechanisms: (1) a lower reduction rate of chlorate due to a lower level of nitrate reductase activity; (2) a lower increase in content of chlorate and/or chlorite and of chloride after chlorate treatment. One mutant of the first type and two mutants of the second type are described. The nitrate reductase-less mutant grows poorly on a medium with nitrate as the only nitrogen source but is not blocked in the uptake of nitrate. Both the other mutants exhibit a nitrate reductase activity equal to or higher than that of the wild type, but probably have a much lowered uptake of chlorate. The latter two mutants belong to the same complementation group, whereas the nitrate reductase-less mutant belongs to a different group.     

Regulation of Nitrate Reductase in Chlorella vulgaris

When excised barley roots (Hordeum distichum L.) are appropriately pretreated, the level of nitrate reductase in the roots increases upon exposure to nitrate. Relatively low levels of nitrate (10 μm) gave maximum induction of nitrate reductase. This increase was inhibited by inhibitors of protein and RNA synthesis, indicating that de novo protein synthesis is probably involved. Induction of nitrate reductase by nitrate is partially prevented by the inclusion of ammonium, an eventual product of nitrate reduction, in the incubation medium. Under the experimental conditions used, ammonium did not inhibit the uptake of nitrate by excised barley roots. It is concluded, therefore, that ammonium, or a product of ammonium metabolism, has a direct effect on the synthesis of nitrate reductase in this tissue.     

Role for nitrate assimilatory genes in virulence of Ustilago maydis

Ustilago maydis can utilize nitrate as a sole source of nitrogen. This process is initiated by transporting nitrate from the extracellular environment into the cell by a nitrate transporter and followed by a two-step reduction of nitrate to ammonium via nitrate reductase and nitrite reductase enzymes, respectively. Here, we characterize the genes encoding nitrate transporter, um03849 and nitrite reductase, um03848 in U. maydis based on their roles in mating and virulence. The deletion mutants for um03848, um03849 or both genes were constructed in mating compatible haploid strains 1/2 and 2/9. In addition, CRISPR-Cas9 gene editing technique was used for um03849 gene to create INDEL mutations in U. maydis mating strains. For all the mutants, phenotypes such as growth ability, mating efficiency and pathogenesis were examined. The growth of all the mutants was diminished when grown in a medium with nitrate as the source of nitrogen. Although no clear effects on haploid filamentation or mating were observed for either single mutant, double Δum03848 Δum03849 mutants showed reduction in mating, but increased filamentation on low ammonium, particularly in the 1/2 background. With respect to pathogenesis on the host, all the mutants showed reduced degrees of disease symptoms. Further, when the deletion mutants were paired with wild type of opposite mating-type, reduced virulence was observed, in a manner specific to the genetic background of the mutant's progenitor. This background specific reduction of plant pathogenicity was correlated with differential expression of genes for the mating program in U. maydis.     

Physiological and biochemical characterization of chlorate-resistant mutants ofAnabaena doliolum

Chlorate-resistant mutants of the filamentous cyanobacterium,Anabaena doliolum, were isolated by N-methyl-N-nitro-N-nitrosoguanidine (MNNG)¹ mutagenesis. Three classes of mutants were obtained that were altered either in the nitrate uptake activity or nitrate reductase enzyme activity or both. These results suggest that the genetic determinant of the uptake system was distinct from that of the reductase system.Uptake studies of nitrite and ammonium and rate of nitrite reductase activity in the mutants revealed that the nitrite and ammonium metabolisms were not affected by this mutation.Both nitrate and chlorate acted like a pair of antagonists, with nitrate protecting the growth against chlorate with increase in its concentration; similarly, increasing chlorate concentrations counteracted the growth-protective action of nitrate.     

Chlorate Toxicity and Nitrate Reductase Activity in Tomato Plants

Chlorate damage was studied in tomato plants ( Lycopersicum esculentum cv. Moneymaker) that were supplied with a nitrogen-free nutrient solution or with a nutrient solution, containing either nitrate or ammonium as a nitrogen source. Damage was low in ammonium-fed plants and high in nitrate-fed plants and in nitrogen-less plants. Nitrate reductase activity could be detected in all treatments, although the activity was highest in the nitrate-fed plants.
The hypothesis that chlorate can be used as a substrate by the enzyme nitrate reductase in higher plants, was studied and proved to be true for the tomato plants, as was found earlier for Escherichia and Chlorella . The affinity of the enzyme for chlorate was lower than for nitrate, the K _m being 4 m M and 0.15 m M respectively. Induction of the enzyme by chlorate could not be detected. The enzyme activity was lowered in leaf discs after a 7 h treatment with chlorate and the inhibition was proportional to the chlorate concentration of the medium.
The results were discussed in terms of competition between nitrate and chlorate at the uptake and the enzyme site and with regard to a possible influence of chlorate on synthesis and breakdown of the enzyme.     

Nitrogen Assimilation in Barley (Hordeum vulgare L. cv. Mazurka) in Response to Nitrate and Ammonium Nutrition

Barley plants (Hordeum vulgare L. cv. Mazurka) were grown inaerated solution cultures with 2 mM or 8 mM inorganic nitrogensupplied as nitrate alone, ammonium alone or 1:1 nitrate+ammonium.Activities of the principal inorganic nitrogen assimilatoryenzymes and nitrogen transport were measured. Activities ofnitrate and nitrite reductases, glutamine synthetase and glutamatesynthase were greater in leaves than in roots but glutamatedehydrogenase was most active in roots. Only nitrate and nitritereductases changed notably (4–10 times) in response tothe different nitrogen treatments. Nitrate reductase appearedto be rate-limiting for nitrate assimilation to glutamate inroots and also in leaves, where its total in vitro activitywas closely related to nitrate flux in the xylem sap and wasslightly in excess of that needed to reduce the transportednitrate. Xylem nitrate concentration was 13 times greater thanthat in the nutrient solution. Ammonium nitrogen was assimilatedalmost completely in the roots and the small amount releasedinto the xylem sap was similar for the nitrate and the ammoniumtreatments. The presence of ammonium in the nutrient decreasedboth export of nitrate to the xylem and its accumulation inleaves and roots. Nitrate was stored in stem bases and was releasedto the xylem and thence to the leaves during nitrogen starvation.In these experiments, ammonium was assimilated principally inthe roots and nitrate in the leaves. Any advantage of this divisionof function may depend partly on total conversion of inorganicnitrogen to amino acids when nitrate and ammonium are givenin optimal concentrations. Hordeum vulgare L., barley, nitrate, ammonium, nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, nitrogen transport