Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Probing the role of copper in the biosynthesis of the molybdenum cofactor in <Emphasis Type=Italic>Escherichia coli</Emphasis> and <Emphasis Type=Italic>Rhodobacter sphaeroides</Emphasis>

The crystal structure of Cnx1G, an enzyme involved in the biosynthesis of the molybdenum cofactor (Moco) in Arabidopsis thaliana, revealed the remarkable feature of a copper ion bound to the dithiolene unit of a molybdopterin intermediate (Kuper et al. Nature 430:803-806, 2004). To characterize further the role of copper in Moco biosynthesis, we examined the in vivo and/or in vitro activity of two Moco-dependent enzymes, dimethyl sulfoxide reductase (DMSOR) and nitrate reductase (NR), from cells grown under a variety of copper conditions. We found the activities of DMSOR and NR were not affected when copper was depleted from the media of either Escherichia coli or Rhodobacter sphaeroides. These data suggest that while copper may be utilized during Moco biosynthesis when it is available, copper does not appear to be strictly required for Moco biosynthesis in these two organisms.     

Identification by mutational analysis of four critical residues in the molybdenum cofactor domain of eukaryotic nitrate reductase

The nucleotide sequence of the nitrate reductase (NR) molybdenum cofactor (MoCo) domain was determined in four Nicotiana plumbaginifolia mutants affected in the NR apoenzyme gene. In each case, missense mutations were found in the MoCo domain which affected amino acids that were conserved not only among eukaryotic NRs but also in animal sulfite oxidase sequences. Moreover an abnormal NR molecular mass was observed in three mutants, suggesting that the integrity of the MoCo domain is essential for a proper assembly of holo-NR. These data allowed to pinpoint critical residues in the NR MoCo domain necessary for the enzyme activity but also important for its quaternary structure.     

Molybdopterin guanine dinucleotide is the organic moiety of the molybdenum cofactor in respiratory nitrate reductase from Pseudomonas stutzeri

Abstract Respiratory nitrate reductase from the denitrifying bacterium Pseudomonas stutzeri is an iron-sulfur enzyme containing the molybdenum cofactor. Hydrolysis of native nitrate reductase with aqueous sulfuric acid revealed 0.92 mol of 5'-GMP per mol of enzyme. The pterin present in the molybdenum cofactor was liberated from the protein and reacted with iodoacetamide. The resulting di(carboxamidomethyl) (cam) derivative was purified on a C₁₈-cartridge and analyzed for its structural elements. Treatment of the cam derivative with nucleotide pyrophosphatase and subsequent HPLC analysis revealed the formation of di(cam)molybdopterin and 5'-GMP at a 1:1 molar ratio and with a yield of 79% with respect to the molybdenum content of the enzyme. Treatment of the cam derivative with nucleotide pyrophosphatase and alkaline phosphatase led to the liberation of 0.51 mol dephosphodi(cam)molybdopterin and of 0.59 mol guanosine per mol of enzyme, which is equal to a molar ratio of 1:2.2. The results indicate, that the organic moiety of the molybdenum cofactor of nitrate reductase from P. stutzeri is molybdopterin guanine dinucleotide of which one mol is contained per mol of nitrate reductase.     

Biochemical genetics of further chlorate resistant, molybdenum cofactor defective, conditional-lethal mutants of barley

Summary Three plants, R9201 and R11301 (from cv. Maris Mink) and R12202 (from cv. Golden Promise), were selected by screening M₂ populations of barley (Hordeum vulgare L.) seedlings (mutagenised with azide in the M₁) for resistance to 10 mM potassium chlorate. Selections R9201 and R11301 were crossed with the wild-type cv. Maris Mink and analysis of the F₂ progeny showed that one quarter lacked shoot nitrate reductase activity. These F₂ plants also withered and died in the continuous presence of nitrate as sole nitrogen source. Loss of nitrate reductase activity and withering and death were due in each case to a recessive mutation in a single nuclear gene. All F1 progeny derived from selfing selection R12202 lacked shoot nitrate reductase activity and also withered and subsequently died when maintained in the continuous presence of nitrate as sole nitrogen source. All homozygous mutant plants lacked not only shoot nitrate reductase activity but also shoot xanthine dehydrogenase activity. The plants took up nitrate, and possessed wild-type or higher levels of shoot nitrite reductase activity and NADH-cytochrome c reductase activity when treated with nitrate for 18 h. We conclude that loss of shoot nitrate reductase activity, xanthine dehydrogenase activity and withering and death, in the three mutants R9201, R11301 and R12202 is due to a mutation affecting the formation of a functional molybdenum cofactor. The mutants possessed wild-type levels of molybdenum and growth in the presence of unphysiologically high levels of molybdate did not restore shoot nitrate reductase or xanthine dehydrogenase activity. The shoot molybdenum cofactor of R9201 and of R12202 is unable to reconstitute NADPH nitrate reductase activity from extracts of the Neurospora crassa nit-1 mutant and dimerise the nitrate reductase subunits present in the respective barley mutant. The shoot molybdenum cofactor of R11301 is able to effect dimerisation of the R11301 nitrate reductase subunits and can reconstitute NADPH-nitrate reductase activity up to 40% of the wild-type molybdenum cofactor levels. The molybdenum cofactor of the roots of R9201 and R11301 is also defective. Genetic analysis demonstrated that R9201, but not R11301, is allelic to R9401 and Az34 (nar-2a), two mutants previously shown to be defective in synthesis of molybdenum cofactor. The mutations in R9401 and R9201 gave partial complementation of the nar-2a gene such that heterozygotes had higher levels of extractable nitrate reductase activity than the homozygous mutants.We conclude that: (a) the nar-2 gene locus encodes a step in molybdopterin biosynthesis; (b) the mutant R11301 represents a further locus involved in the synthesis of a functional molybdenum cofactor; (c) mutant Rl2202 is also defective in molybdopterin biosynthesis; and (d) the nar-2 gene locus and the gene locus defined by R11301 govern molybdenum cofactor biosynthesis in both shoot and root.     

Different forms of molybdenum cofactor inVicia faba seeds: The presence of molybdenum cofactor carrier protein and its purification

There were significant differences in the contents of molybdenum cofactor (Mo-co), both in a low-molecular-mass form (free Mo-co) and in a protein-bound form, in seeds of sevenVicia faba genotypes. Low-molecular-mass Mo-co species present in the extracts were detected by their ability to reactivate, through a dialysis membrane, aponitrate reductase from theNeurospora crassa nit-1 mutant. In extracts of all genotypes tested, the amount of Mo-co capable of directly reactivating nitrate reductase of theN. crassa nit-1 mutant was always much higher than that of low-molecular-mass Moco. These data cannot be explained by considering, as traditionally, that Mo-co detected directly, i.e. without any previous treatment for its release from Mo-coproteins, corresponds to free low-molecular mass Mo-co. A protein which bound Mo-co was purified to electrophoretic homogeneity. This protein consisted of a single 70-kDa polypeptide chain and carried a Mo-co that could be efficiently released when in contact with aponitrate reductase.Abbreviations CP carrier protein - Mo-co molybdenum cofactor - NR nitrate reductase - XO xanthine oxidase     

Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

Summary The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant ofArabidopsis thaliana, ch12, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained.chl2 plants have low levels of NR activity, yet the map position of thechl2 mutation is clearly distinct from that of the two NR structural genes that have been identified inArabidopsis. In addition,chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest thatchl2 may be a NR regulatory mutant. We have examinedchl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels inchl2 plants using a sensitive, specific assay for MoCo: complementation ofNeurospora MoCo mutant extracts. We found thatchl2 has low levels of MoCo — about one-eighth the wild-type level and less than the level in anotherArabidopsis MoCo mutantchl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Bothchl2 andchl6 are sensitive to tungstate at concentrations that have no effect on wildtype plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to thechl2 locus, 10 centimorgans fromerecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant ofArabidopsis, ch17, which is a MoCo mutant with the same phenotypes aschl2 andchl6.     

Genetic analysis of nitrate reductase deficient mutants of Nicotiana plumbaginifolia: Evidence for six complementation groups among 70 classified molybdenum cofactor deficient mutants

Summary A total of 70 cnx mutants have been characterized from a collection of 211 nitrate reductase deficient (NR^-) mutants isolated from mutagenized Nicotiana plumbaginifolia protoplast cultures after chlorate selection and regeneration into plants. They are presumed to be affected in the biosynthesis of the molybdenum cofactor since they are also deficient for xanthine dehydrogenase activity but contain NR apoenzyme. The remaining clones were classified as nia mutants. Sexual crosses performed between cnx mutants allowed them to be classified into six independent complementation groups. Mutants representative of these complementation groups were used for somatic hybridization experiments with the already characterized N. plumbaginifolia mutants NX1, NX24, NX23 and CNX103 belonging to the complementation groups cnxA, B, C and D respectively. On the basis of genetic analysis and somatic hybridization experiments, two new complementation groups, cnxE and F, not previously described in higher plants, were characterized. Unphysiologically high levels of molybdate can restore the NR activity of cnxA mutant seedlings in vivo, but cannot restore NR activity to any mutant from the other cnx complementation groups.     

The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon Haloferax mediterranei

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO₃⁻, NO₂⁻ or NH₄⁺) for growth. Different approaches were considered to study the effect of NH₄⁺ on nitrogen assimilation in Hfx. mediterranei cells grown in KNO₃ medium. The NH₄⁺ addition to KNO₃ medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH₄⁺ media. Both activities increased when NH₄⁺ was removed from culture, showing that the negative effect of NH₄⁺ on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO₃⁻ and NH₄⁺, NH₄⁺ was preferentially consumed, but NO₃⁻ uptake was not completely inhibited by NH₄⁺ at prolonged time scale. The addition of MSX to NH₄⁺ or NO₃⁻ cultures results in an increase of Nas and NiR activities, suggesting that NH₄⁺ assimilation, rather than NH₄⁺ per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient in nitrate respiration

A Paracoccus denitrificans strain (M6Ω) unable to use nitrate as a terminal electron acceptor was constructed by insertional inactivation of the periplasmic and membrane-bound nitrate reductases. The mutant strain was able to grow aerobically with nitrate as the sole nitrogen source. It also grew anaerobically with nitrate as sole nitrogen source when nitrous oxide was provided as a respiratory electron acceptor. These growth characteristics are attributed to the presence of a third, assimilatory nitrate reductase. Nitrate reductase activity was detectable in intact cells and soluble fractions using nonphysiological electron donors. The enzyme activity was not detectable when ammonium was included in the growth medium. The results provide an unequivocal demonstration that P. denitrificans can express an assimilatory nitrate reductase in addition to the well-characterised periplasmic and membrane-bound nitrate reductases. Received: 12 August 1996 / Accepted: 29 October 1996     

Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol [NO(3)(-)] min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol [NO(3)(-)] min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at >20 degrees C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Characterization of the molybdate transport system ModABC of Staphylococcus carnosus

Transposon mutagenesis of Staphylococcus carnosus led to the identification of three genes, modABC, which encode an ABC transporter that is involved in molybdate transport. It was shown by [¹⁴C]palmitate labeling that ModA represents a lipoprotein that in gram-positive bacteria is the counterpart of the periplasmic binding proteins of gram-negative organisms. The sequence characteristics identify ModB as the integral-membrane, channel-forming protein and ModC as the ATP-binding energizer for the transport system. Mutants defective in modABC had only 0.4% of the wild-type nitrate reductase activity. Molybdate at a non-physiologically high concentration (100 μM) fully restored nitrate reductase activity, suggesting that at least one other system is able to transport molybdate, but with lower affinity. The expression of modA (and most likely of modBC) was independent of oxygen and nitrate. To date, there are no indications for molybdate-specific regulation of modABC expression since in a modB mutant, modA expression was unchanged in comparison to the wild-type. Received: 5 February 1999 / Accepted: 31 May 1999     

Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC7942

Summary The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1–254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O₂ evolution (photosynthetic O₂ evolution coupled to nitrate reduction) in wild-type cells measured in the presence of l-methionine d,l-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 M), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O₂ evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system.     

Partitioning of inorganic nitrogen assimilation between the roots and shoots of cerrado and forest trees of contrasting plant communities of South East Brasil

Summary Woody plants growing in cerrado and forest communities of south-east Brasil were found to have low levels of nitrate reductase activity in their leaves suggesting that nitrate ions are not an important nitrogen source in these communities. Only in the leaves of species growing in areas of disturbance, such as gaps and forest margins, were high levels of nitrate reductase present. When pot-grown plants were supplied with nitrate, leaves and roots of almost all species responded by inducing increased levels of nitrate reductase. Pioneer or colonizing species exhibited highest levels of nitrate reductase and high shoot: root nitrate reductase activities. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase were present in leaves and roots of the species examined.¹⁵N-labelled nitrate and ammonium were used to compare the assimilatory characteristics of two species:Enterolobium contortisiliquum, with a high capacity to reduce nitrate, andCalophyllum brasiliense, of low capacity. The rate of nitrate assimilation in the former was five times that of the latter. Both species had similar rates of ammonium assimilation. Results for eight species of contrasting habitats showed that leaf nitrogen content increased in parallel with xylem sap nitrogen concentrations, suggesting that the ability of the root system to acquire, assimilate or export nitrate determines shoot nitrogen status. These results emphasise the importance of nitrogen transport and metabolism in roots as determinants of whole plant nitrogen status.     

Expression and characterization of the assimilatory NADH-nitrite reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

A nas gene region from Rhodobacter capsulatus E1F1 containing the putative nasB gene for nitrite reductase was previously cloned. The recombinant His₆-NasB protein overproduced in E. coli showed nitrite reductase activity in vitro with both reduced methyl viologen and NADH as electron donors. The apparent K _m values for nitrite and NADH were 0.5 mM and 20 μM, respectively, at the pH and temperature optima (pH 9 and 30°C). The optical spectrum showed features that indicate the presence of FAD, iron-sulfur cluster and siroheme as prosthetic groups, and nitrite reductase activity was inhibited by sulfide and iron reagents. These results indicate that the phototrophic bacterium R. capsulatus E1F1 possesses an assimilatory NADH-nitrite reductase similar to that described in non-phototrophic organisms.     

Isolation of cDNA clones coding for spinach nitrite reductase: Complete sequence and nitrate induction

Summary The main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclearencoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.