Regulation Of Nitrate Assimilation Research Articles

Growth and Nitrate Reductase Activity Are Impaired in Rice Osnlp4 Mutants Supplied with Nitrate.

Nitrate is an important nutrient and signaling molecule in plants, which modulates the expression of many genes and regulates plant growth. In paddy-grown rice (Oryza sativa), nitrogen is mostly supplied in the form of ammonium but can also be supplied in the form of nitrate. Several nitrogen transporters and nitrate assimilation enzymes have been identified and functionally characterized in rice. However, little is known regarding the nitrate sensing system in rice, and the regulatory mechanisms of nitrate-related genes remain to be elucidated. In recent years, NIN-like proteins (NLPs) have been described as key transcription factors of nitrogen responses in Arabidopsis thaliana, which implies that OsNLP4 is involved in the regulation of nitrate assimilation and nitrogen use efficiency in rice. Here, we show that OsNLP4 can influence plant growth by affecting nitrate reductase (NR) activity. The growth of OsNLP4 knockdown mutants was reduced when nitrate was supplied, but not when ammonium was supplied. The nitrate concentration was significantly reduced in osnlp4 mutants. Furthermore, the concentrations of iron and molybdenum, essential elements for NR activity, were reduced in OsNLP4 knockdown mutants. We propose that, in addition to the regulation of gene expression within the nitrate signaling pathway, OsNLP4 can affect the NR activity and nitrate-dependent growth of rice. Our results support a working model for the role of OsNLP4 in the nitrate signaling pathway.

Understanding nitrate assimilation and its regulation in microalgae.

Nitrate assimilation is a key process for nitrogen (N) acquisition in green microalgae. Among Chlorophyte algae, Chlamydomonas reinhardtii has resulted to be a good model system to unravel important facts of this process, and has provided important insights for agriculturally relevant plants. In this work, the recent findings on nitrate transport, nitrate reduction and the regulation of nitrate assimilation are presented in this and several other algae. Latest data have shown nitric oxide (NO) as an important signal molecule in the transcriptional and posttranslational regulation of nitrate reductase and inorganic N transport. Participation of regulatory genes and proteins in positive and negative signaling of the pathway and the mechanisms involved in the regulation of nitrate assimilation, as well as those involved in Molybdenum cofactor synthesis required to nitrate assimilation, are critically reviewed.

Bacterial nitrate assimilation: gene distribution and regulation

In the context of the global nitrogen cycle, the importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognized. In contrast, the utilization of nitrate by heterotrophic bacteria has historically received less attention because the primary role of these organisms has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs. Until now, regulation of nitrate assimilation has been mainly studied in cyanobacteria. In contrast, in heterotrophic bacterial strains, the study of nitrate assimilation regulation has been limited to Rhodobacter capsulatus, Klebsiella oxytoca, Azotobacter vinelandii and Bacillus subtilis. In Gram-negative bacteria, the nas genes are subjected to dual control: ammonia repression by the general nitrogen regulatory (Ntr) system and specific nitrate or nitrite induction. The Ntr system is widely distributed in bacteria, whereas the nitrate/nitrite-specific control is variable depending on the organism.

Regulation of nitrate assimilation in cyanobacteria

Nitrate assimilation by cyanobacteria is inhibited by the presence of ammonium in the growth medium. Both nitrate uptake and transcription of the nitrate assimilatory genes are regulated. The major intracellular signal for the regulation is, however, not ammonium or glutamine, but 2-oxoglutarate (2-OG), whose concentration changes according to the change in cellular C/N balance. When nitrogen is limiting growth, accumulation of 2-OG activates the transcription factor NtcA to induce transcription of the nitrate assimilation genes. Ammonium inhibits transcription by quickly depleting the 2-OG pool through its metabolism via the glutamine synthetase/glutamate synthase cycle. The P(II) protein inhibits the ABC-type nitrate transporter, and also nitrate reductase in some strains, by an unknown mechanism(s) when the cellular 2-OG level is low. Upon nitrogen limitation, 2-OG binds to P(II) to prevent the protein from inhibiting nitrate assimilation. A pathway-specific transcriptional regulator NtcB activates the nitrate assimilation genes in response to nitrite, either added to the medium or generated intracellularly by nitrate reduction. It plays an important role in selective activation of the nitrate assimilation pathway during growth under a limited supply of nitrate. P(II) was recently shown to regulate the activity of NtcA negatively by binding to PipX, a small coactivator protein of NtcA. On the basis of accumulating genome information from a variety of cyanobacteria and the molecular genetic data obtained from the representative strains, common features and group- or species-specific characteristics of the response of cyanobacteria to nitrogen is summarized and discussed in terms of ecophysiological significance.

Identification and characterization of the two-component NtrY/NtrX regulatory system in Azospirillum brasilense.

Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans. These A. brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized. The present study suggests that ORF1 and ORF2 correspond to the A. brasilense ntrY and ntrX genes, respectively. The amino acid sequences of A. brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively. Analysis of lacZ transcriptional fusions by the beta-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A. brasilense. The ntrYX operon complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth, suggesting a possible cross-talk between the NtrY/X and NtrB/C sensor/regulator pairs. Our data support the existence of another two-component regulatory system in A. brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation.

Regulation of nitrate assimilation in the cyanobacterium Phormidium laminosum

The intracellular ratio of 2-oxoglutarate to glutamine has been analyzed under nutritional conditions leading to different activity levels of nitrate-assimilating enzymes in Phormidium laminosum (Agardh) Gom. This non-N2-fixing cyanobacterium adapted to the available nitrogen source by modifying its nitrate reductase (NR; EC 1.7.7.2), nitrite reductase (NiR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) activities. The 2-oxoglutarate/glutamine ratio was similar in cells adapted to grow with nitrate or ammonium. However, metabolic conditions that increased this ratio [i.e., nitrogen starvation or l-methionine-d,l-sulfoximine (MSX) treatment] corresponded to high activity levels of NR, NiR, GS (except in MSX-treated cells) and glutamate synthase (GOGAT; EC 1.4.7.1). By contrast, metabolic conditions that diminished this ratio (i.e., addition of ammonium to nitrate-growing cells or addition of nitrate or ammonium to nitrogen-starved cells) resulted in low activity levels. The variation in the 2-oxoglutarate/glutamine ratio preceded the changes in enzyme activities. These results suggest that changes in the 2-oxoglutarate/glutamine ratio could be the signal that triggers the adaptation of P. laminosum cells to variations in the available nitrogen source, as occurs in enterobacteria.

Primary nitrogen assimilation in higher plants and its regulation

Characteristics of the enzymes involved in the assimilation of NO3− and NH4+, in particular the nitrate and nitrite reductases, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, glutamate decarboxylase, and asparagine synthetase, are described. The cellular organization of these enzymes in root and leaf tissues are assessed in view of recent research developments that utilize various tissue blotting techniques. Regulation of nitrate assimilation is analyzed at the physiological, biochemical, and molecular levels. Key words: nitrate, ammonium, assimilation, regulation.

An amino-acid-grown maize cell line for use in investigating nitrate assimilation

Studies on uptake and assimilation of nitrate in plants are confounded by differences in cell function associated with anatomical features of roots as well as by problems inherent with growing plants without nitrate. To circumvent these problems, a Zea mays L. embryo cell line was grown in suspension culture using an amino-acid-based medium consisting of a Murashige and Skoog medium in which ammonium and nitrate were replaced by aspartic acid (100 mg/l), glycine (100 mg/l), arginine (150 mg/l), and glutamine (1 g/l). The growth, cellular characteristics, and physical appearance of the amino-acid-grown cells were similar to cells grown in the presence of nitrate. The amino-acid-grown cells exhibited the expected induction pattern and inhibitor sensitivity of nitrate uptake. This cell line should facilitate research on the induction of nitrate uptake and the regulation of nitrate assimilation into proteins.

Zea3: a pleiotropic mutation affecting cotyledon development, cytokinin resistance and carbon-nitrogen metabolism

When photomorphogenesis takes place during early plant development, the cotyledons undergo a metabolic transition from heterotrophic sink metabolism to autotrophic source metabolism. A mutant screen was devised for seedlings affected in the regulation of nitrate assimilation during this early sink-source transition in Nicotiana plumbaginifolia. A mutant (EMS 203.6) was isolated for its inability to grow on low nitrate concentration. In contrast to wild-type (WT) plants, the mutant cotyledons remained tightly attached to each other throughout seedling development. It was found that a low carbon/nitrogen ratio (C/N ratio) in the medium was required for mutant growth. The higher the ratio was, the more the growth was inhibited. Mutant EMS 203.6 accumulated all amino acids in permissive conditions (low C/N ratio), and all amino acids and sugars also in selective (high C/N ratio) conditions. In addition, sucrose in the medium repressed light-regulated genes involved in nitrate assimilation and in photosynthesis in the mutant but not in the WT plants. The mutation was mapped to the Zea3 complementation group which confers resistance to zeatin. This zeatin resistance was associated with a hypertrophy of mutant cotyledons in response to cytokinin. Both cytokinin resistance and sensitivity to a high C/N ratio were not observed in etiolated mutant seedlings and were restricted to the jointed-cotyledon developmental stage. Previous physiological studies showed evidence for a role of cytokinins in the expression of nitrate reductase. Here, the first genetic evidence for a link between carbohydrate/nitrogen metabolism and cytokinin action during early development is provided.

Regulation of Nitrate Assimilation in Plants—A Metabolic Perspective

Regulation of Nitrate Assimilation in Plants—A Metabolic Perspective

Effect of UVB irradiation on enzymes of nitrogen metabolism in turions of Spirodela polyrhiza (L.) Schleiden

The influence of UVB irradiation on the enzymes of nitrogen metabolism (nitrate reductase, EC 1.6.6.1, nitrite reductase, EC 1.7.7.1, alanine aminotransferase, EC 2.6.1.2, aspartate aminotransferase, EC 2.6.1.1, glutamine synthetase, EC 6.3.1.2) and the uptake of ammonium and nitrate in turions of Spirodela polyrhiza was investigated. UVB irradiation was started either immediately after transferring the turions from 5 °C (after-ripening conditions) to 25 °C (germination conditions) or after a storage period at 25 °C in darkness for 72 h. The activities of all enzymes investigated were decreased by UVB irradiation (12 or 24 h). The activity of nitrate reductase was more strongly inhibited than that of nitrite reductase, and the activity of alanine aminotransferase was more strongly decreased than that of aspartate aminotransferase. The inhibitory effect of UVB on glutamine synthetase was only small. Within a period of 5 h neither the uptake of ammonium nor that of nitrate was influenced by UVB irradiation. Because UVB irradiation did not influence the light-dependent germination of turions, the results presented support the conclusion that the regulation of germination and the regulation of nitrate assimilation are unrelated phenomena in turions

Effect of Nitrogen Supply on the Kinetics and Regulation of Nitrate Assimilation in Chlamydomonas reinhardtii Dangeard

Concentration-dependent NO3 uptake by C. reinhardtii was studied in cells cultured under three NO3-growth regimes: 10 mM NO3 (NOJ -grown), 0-25 mM NO3 (N-limited) and an 18 h period of N-deprivation following growth on 10 mM NO J (N-starved). Two NO3 uptake isotherms, with an apparent phase transition in uptake at IT mM NO3, were resolved in all three cell types over the concentration range tested (0025 to 1-8 mM N03). The apparent Kmax for uptake by N-limited cells (0-72 x 10-8 /.?mol cell-1 h-1) exceeded that for both NO3-grown (0-42 x 10-8 /??mol cell-1 h-1) and N-starved (0-49 x 10-8 /??mol cell-1 h-1) cells at NO3 concentrations less than IT mM (isotherm 0). When supplied with NOj concentrations in excess of IT mM (isotherm 1) the apparent Kmax values for uptake by N-limited (0-64 x 10-8 /?mol cell-1 h-1) and N-starved (0-41 x 10-8 /?mol cell-1 h-1) cells were depressed relative to those for isotherm 0. In contrast, NO3-grown cells exhibited a greater apparent Vmax for uptake (0-63 x 10-8/?mol cell-1 h-1) at the higher NO3 concentrations. When N-limited and N-starved cells were supplied with 1-6 mM NO J (isotherm 1), the pattern of uptake showed a rapid linear increase after 60 min. This enhanced pattern of uptake did not occur in NO J-grown cells supplied with either 0-6 or 1-6 mM NO3 or in N-limited and N-starved cells supplied with 0-6 mM NO J. Cycloheximide (10 /?g cm-3), but not actinomycin D (50 /?g cm-3), blocked the apparent 'induction' of NO3 uptake in N-starved cells supplied with l-6mM NO J. These results suggest that mechanisms controlling the observed 'inducible' aspect of NO3 uptake in N-starved cells are dependent on a translational or post translational event. It is possible that the NO3 -concentration dependent 'inducible' portion of uptake may provide the means by which C. reinhardtii cells may regulate uptake in accordance with their capacity for assimilation.

Isolation of a gene that down-regulates nitrate assimilation and influences another regulatory gene in the same system.

Glutamine is the preferred source of nitrogen of Neurospora crassa. In its presence and that of the gene product of MS5 (nmr-1), the fungus represses the assimilation of less preferred forms of nitrogen, such as nitrate. In the absence of glutamine and the presence of the product of gene nit-2, less preferred forms of nitrogen are assimilated as long as a specific pathway for their assimilation is induced. We report here the isolation, from a cosmid bank, of a gene that complements MS5 and can also complement nit-2. We speculate that this result suggests an interaction between the MS5 and nit-2 gene products and that this is important in the regulation of nitrate assimilation.

Isolation of a Gene That Down-Regulates Nitrate Assimilation and Influences Another Regulatory Gene in the Same System

Glutamine is the preferred source of nitrogen of Neurospora crassa. In its presence and that of the gene product of MS5 (nmr-1), the fungus represses the assimilation of less preferred forms of nitrogen, such as nitrate. In the absence of glutamine and the presence of the product of gene nit-2, less preferred forms of nitrogen are assimilated as long as a specific pathway for their assimilation is induced. We report here the isolation, from a cosmid bank, of a gene that complements MS5 and can also complement nit-2. We speculate that this result suggests an interaction between the MS5 and nit-2 gene products and that this is important in the regulation of nitrate assimilation.

Regulation of nitrate assimilation by ammonium in soils and in isolated soil microorganisms

The regulation of NO − 3 assimilation by NH + 4 and amino acids was studied to better define the conditions under which raicrobial NO − 3 assimilation could be expected to take place in nature. The assay system was microbial isolates from soil and slurries of two soils enhanced in NO − 3 assimilating capacity by previous exposure to glucose for 18–24 h. The NO − 3 assimilation rate was monitored by a NO − 3 electrode and the inhibition determined by the change in rate following addition of NH + 4 or amino acids. NH + 4 inhibited (by 60%) NO − 3 assimilation immediately (< 1 min) and at very low NH + 4 concentrations (O.1 μg N g −1 soil). At higher NH + 4 concentrations (10 μg N g − soil) a second state of inhibition (80%) occurred after 5 min. The speed of the first inhibition stage suggests that the site of inhibition is the transport of NO − 3 into the cell. Asparagine was the only amino acid to act as rapidly and effectively as NH + 4 in the soil, and thus the amino acids are unlikely to be important regulators of NO − 3 assimilation in soils. Of the three soil isolates studied, Saccharomyces cerevisiae and Pseudomonas fluorescens responded to NH + 4 and amino acids in a manner similar to the soil but NO − 3 assimilation in the third organism, Azotobacter vinelandii, was generally insensitive to these N substrates in this short-term assay. Only high concentrations of NH + 4 caused a partial inhibition of NO − 3 assimilation in A. vinelandii. Due to the effectiveness of low NH + 4 concentrations in inhibiting NO − 3 assimilation in soils, microbial immobilization is not expected to be an important fate of NO − 3 unless there are NH + 4-free microsites in the soil.

Mutants of Azotobacter vinelandii altered in the regulation of nitrate assimilation

The two enzymes involved in the assimilatory pathway of nitrate in Azotobacter vinelandii are corregulated. Nitrate reductase and nitrite reductase are inducible by nitrate and nitrite. Ammonium represses induction by nitrate of both reductases. Repression by ammonium is higher in media containing 2-oxo-glutarate as carbon source than in media containing sucrose. Mutants in the gene ntrC lost nitrate and nitrite reductase simultaneously. Ten chlorate-resistant mutants with a new phenotype were isolated. In media without ammonium they had a normal phenotype, being sensitive to the toxic effect of chlorate. In media containing low ammonium concentrations they were resistant to chlorate. These mutants seem to be affected in the repression of nitrate and nitrite reductases by ammonium.

Regulation of nitrate assimilation in plants in light and dark

Assimilation of nitrate and nitrite accumulated in leaves was much more rapid in the light than in the dark. Under light aerobic and dark anaerobic conditions the extent of nitrate reduction was almost equal. Dark assimilation of nitrite was drastically inhibited by 2,4-dinitrophenol (2,4-DNP). Although the uncoupler did not show any significant effect on oxidation of NADH and succinate in isolated leaf mitochondria, it inhibited 14CO 2 evolved from endogenously labelled sugars, probably by blocking the formation of glucose 6-phosphate (G 6-P). It is suggested that for dark assimilation of nitrite, NADPH generated in the oxidative pentose phosphate pathway is the source of reductant.

Light mediated regulation of nitrate assimilation in Synechococcus leopoliensis

Synechococcus leopoliensis was cultivated in a light/dark regime of 12:12 h. After onset of the illumination (2 h), the specific activity of nitrite reductase, glutamine synthetase and isocitric dehydrogenase increased; that of glucose-6-phosphate dehydrogenase decreased and that of nitrate reductase and NAD- (NADP) glutamate dehydrogenase remained nearly unchanged.

Effect of ammonium on the regulation of nitrate assimilation in natural phytoplankton populations

The effect of ammonium additions (0.5 to 10 μg-at. N · 1 −1) on the nitrate reductase activity and on the nitrate uptake of natural marine phytoplankton populations was investigated. Specific nitrate reductase activity was significantly reduced at all ammonium concentrations after 24 h of the addition, but not after 6 h. Total nitrate reductase activity did not decrease even at the highest ammonium concentrations. From this it is concluded that the ammonium concentrations used during this study affected mostly the synthesis of new active enzyme. Nitrate uptake rates were also reduced by the addition of ammonium. In contrast to the nitrate reductase results, maximum inhibition was, however, observed after 6 h rather than after 24 h. The lag in the time of response between the inhibition after the ammonium addition suggests 1. (1) that NH 4 + affects nitrate assimilation in two steps, first nitrate uptake and second nitrate reduction; and 2. (2) that the mechanisms controlling these processes are to a certain extent independent of each other.

Effect of ammonium on the regulation of nitrate assimilation in natural phytoplankton populations : Blasco, D. and H.L. Conway, 1982. J. expl mar. Biol. Ecol., 61(2):157–168

Effect of ammonium on the regulation of nitrate assimilation in natural phytoplankton populations : Blasco, D. and H.L. Conway, 1982. J. expl mar. Biol. Ecol., 61(2):157–168