Effect of nodulation on the nitrate assimilation in vegetative soybean plants

Summary Nitrate assimilation in the first trifoliate leaf of vegetative soybean plants (Glycine max L. Merr, cv Hodgson) was studied in relation to nodulation. Nodulated and non-nodulated plants were grown in a nitrate medium (4 mM). As a control nodulated plants were grown in a nutrient medium without combined nitrogen. This study included measurements of the acetylene reduction activity of the whole plant and of thein vitro nitrate reductase, glutamine synthetase and glutamate dehydrogenase activities in the first leaf and of the nitrate concentration. Nitrate accumulation and nitrate reductase activity were depressed in nodulated plants; root growth was decreased in the presence of nitrate. The relationships between nitrate assimilation and nodulation are discussed.     

Isolation and characterization of nitrate reductase deficient mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum

To clarify the role of the fungal nitrate assimilation pathway in nitrate reduction by mycorrhizal plants, nitrate reductase (NR)-deficient (NR⁻) mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi have been selected. These mutants were produced by u.v. mutagenesis on protoplasts originating from homokaryotic mycelia belonging to complementary mating types of this heterothallic tetrapolar species. Chlorate-resistant mutants were first selected in the presence of different nitrogen (N) sources in the culture medium. Among 1495 chlorate resistant mycelia, 30 failed to grow on nitrate and lacked a detectable NR activity. Growth tests on different N sources suggested that the NR activity of all the different mutants is specifically impaired as a result of mutations in either the gene coding for NR apoprotein or genes controlling the synthesis of the molybdenum cofactor. Furthermore, restoration of NR activity in some of the dikaryons obtained after crosses between the different mutant mycelia suggested that not all the selected mutations mapped in the same gene. Utilization of N on a NH₄¹⁵NO₃ medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas ¹⁵N enrichment values indicated that 13–27% of N present in 13-d-old wild-type mycelia originated from nitrate. Apparently, the mutant mycelia do not compensate their inability to use nitrate by a more efficient use of ammonium. These different NR mutants still form mycorrhizas with the habitual host plant, Pinus pinaster (Ait.), making them suitable for study of the contribution of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal plants.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Expression of the Arabidopsis thaliana invertase gene family

Two new loci have been found to be clustered with five other genes for the nitrate assimilation pathway in the Chlamydomonas reinhardtii genome. One gene, located close to the 3′-end of the high-affinity nitrate transporter (HANT) gene Nrt2;2, corresponds to the nitrite reductase (NiR) structural gene Nii1. This is supported by a number of experimental findings: (i) NiR-deficient mutants have lost Nii1 gene expression; (ii) Nii1 mRNA accumulation is co-regulated with the expression of other structural genes of the nitrate assimilation pathway; (iii) nitrite (nitrate) utilization ability is recovered in the NiR mutants by functional complementation with a wild-type Nii1 gene; (iv) the elucidated NII1 amino acid sequence is highly similar to that of the cyanobacterial and higher-plant enzyme, and contains the predicted domains for plastidic ferredoxin-NiRs. Thus, the mutant phenotype and the mRNA sequence and expression of the Nii1 gene have been unequivocally related. Accumulation of mRNA for the second locus identified, Lde1 (light-dependent expression), was not regulated by nitrogen, but like nitrate-assimilation clustered genes, its expression was down-regulated in the dark. Received: 27 November 1997 / Accepted: 19 January 1998     

Molecular approaches to the analysis of nitrate assimilation

Abstract. The application of molecular approaches such as mutant analysis and recombinant DNA technology, in conjunction with immunology, are set to revolutionize our understanding of the nitrate assimilation pathway. Mutant analysis has already led to the identification of genetic loci encoding a functional nitrate reduction step and is expected to lead ultimately to the identification of genes encoding nitrate uptake and nitrite reduction. Of particular significance would be identification of genes whose products contribute to regulatory networks controlling nitrogen metabolism. Recombinant DNA techniques are particularly powerful and have already allowed the molecular cloning of the genes encoding the apoprotein of nitrate reductase and nitrite reductase. These successes allow for the first lime the possibility to study directly the role of environmental factors such as type of nitrogen source (NO₃⁻ or NH₄⁺) available to the plant, light, temperature water potential and CO₂ and O₂ tensions on nitrate assimilation gene expression and its regulation at the molecular level. This is an important advance since our current understanding of the regulation of nitrate assimilation is based largely on changes of activity of the component steps. The availability of mutants, cloned genes, and gene transfer systems will permit attempts to manipulate the nitrate assimilation pathway.     

Methylammonium-resistant mutants ofNicotiana plumbaginifolia are affected in nitrate transport

This work reports the isolation and preliminary characterization ofNicotiana plumbaginifolia mutants resistant to methylammonium.Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up byNicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.     

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.     

Nitrite-responsive activation of the nitrate assimilation operon in Cyanobacteria plays an essential role in up-regulation of nitrate assimilation activities under nitrate-limited growth conditions

NtcB of the cyanobacterium Synechococcus elongatus strain PCC 7942 is a LysR family protein that enhances expression of the nitrate assimilation operon (nirA operon) in response to the presence of nitrite, an intermediate of assimilatory nitrate reduction. Inactivation of ntcB in this cyanobacterium specifically abolishes the nitrite responsiveness of nirA operon expression, but under nitrate-replete conditions (wherein negative feedback by intracellularly generated ammonium prevails over the positive effect of nitrite) activity levels of the nitrate assimilation enzymes are marginally higher in the wild-type cells than in the mutant cells, raising the issue of whether the nitrite-promoted regulation has physiological importance. On the other hand, the strains carrying ntcB expressed much higher nitrate assimilation enzyme activities under nitrate-limited growth conditions than under nitrate-replete conditions whereas the ntcB-deficient strains showed levels of the enzyme activities lower than those seen under the nitrate-replete conditions. Although the ntcB mutant maintained a constant cell population in a nitrate-limited chemostat when grown as a single culture, it was diluted at a rate expected for nondividing cells when mixed with the wild-type cells and subjected to nitrate limitation in the chemostat culture system. These results demonstrated that the nitrite-promoted activation of the nitrate assimilation operon is essential for up-regulation of the nitrate assimilation activities under the conditions of nitrate limitation and for competitive utilization of nitrate.     

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.     

Genomic analysis of the nitrate response using a nitrate reductase-null mutant of Arabidopsis

A nitrate reductase (NR)-null mutant of Arabidopsis was constructed that had a deletion of the major NR gene NIA2 and an insertion in the NIA1 NR gene. This mutant had no detectable NR activity and could not use nitrate as the sole nitrogen source. Starch mobilization was not induced by nitrate in this mutant but was induced by ammonium, indicating that nitrate was not the signal for this process. Microarray analysis of gene expression revealed that 595 genes responded to nitrate (5 mm nitrate for 2 h) in both wild-type and mutant plants. This group of genes was overrepresented most significantly in the functional categories of energy, metabolism, and glycolysis and gluconeogenesis. Because the nitrate response of these genes was NR independent, nitrate and not a downstream metabolite served as the signal. The microarray analysis also revealed that shoots can be as responsive to nitrate as roots, yet there was substantial organ specificity to the nitrate response.