Mechanism and importance of post-translational regulation of nitrate reductase

In higher plants, nitrate reductase (NR) is inactivated by the phosphorylation of a conserved Ser residue and binding of 14-3-3 proteins in the presence of divalent cations or polyamines. A transgenic Nicotiana plumbaginifolia line (S521) has been constructed where the regulatory, conserved Ser 521 of tobacco NR (corresponding to Ser 534 in Arabidopsis) was mutated into Asp. This mutation resulted in the complete abolition of activation/inactivation in response to light/dark transitions or other treatments known to regulate the activation state of NR. Analysis of the transgenic plants showed that, under certain conditions, when whole plants or cut tissues are exposed to high nitrate supply, post-translational regulation is necessary to avoid nitrite accumulation. Abolition of the post-translational regulation of NR also results in an increased flux of nitric oxide from the leaves and roots. In view of the results obtained from examining the different transgenic N. plumbaginifolia lines, compartmentation of nitrate into an active metabolic pool and a large storage pool appears to be an important factor for regulating nitrate reduction. The complex regulation of nitrate reduction is likely to have evolved not only to optimize nitrogen assimilation, but also to prevent and control the formation of toxic, and possibly regulatory, products of NR activities. Phos phorylation of NR has previously been found to influence the degradation of NR in spinach leaves and Arabidopsis cell cultures. However, experiments with whole plants of N. plumbaginifolia, Arabidopsis, or squash are in favour of NR degradation being the same in light and darkness and independent of phosphorylation at the regulatory Ser.     

Amino Acid interactions in the regulation of nitrate reductase induction in cotton root tips

Glycine, asparagine, and glutamine inhibited the induction by nitrate of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.). This inhibition was partially or entirely prevented when the inhibitor was applied in combination with any of several other amino acids. Studies of ¹⁴C-labeled amino acid uptake showed that, in most cases, the apparent antagonism resulted simply from competition for uptake. However, certain antagonists did not curtail uptake. The most effective of these were leucine (against all three inhibitors), and isoleucine and valine (against asparagine or glutamine, but not glycine). These results show that interactions among amino acids in the regulation of nitrate reductase induction result from at least two mechanisms, one acting on uptake of inhibitory amino acids, and the other involving true antagonism.     

Sulfur starvation and restoration affect nitrate uptake and assimilation in rapeseed

We analyzed the effect of omission of sulfur (S) from the nutrient solution and then restoration of S-source on the uptake and assimilation of nitrate in rapeseed. Incubation in nutrient solution without S for 1–6 days led to decline in uptake of nitrate, activities, and expression levels of nitrate reductase (NR) and glutamine synthetase (GS). The nitrite reductase (NiR) and glutamate synthase (GOGAT) activities were not considerably affected. There was significant enhancement in nitrate content and decline in sulfate content. Evaluation of amino acid profile under S-starvation conditions showed two- to fourfold enhancement in the contents of arginine, asparagine and O-acetyl-l-serine (OAS), whereas the contents of cysteine and methionine were reduced heavily. When the S-starved plants were subjected to restoration of S for 1, 3, 5, and 7 days, activities and expression levels of NR and GS recovered within the fifth and seventh days of restoration, respectively. Exogenous supply of metabolites (arginine, asparagine, cysteine, glutamine, OAS, and methionine) also affected the uptake and assimilation of nitrate, with a maximum for OAS. These results corroborate the tight interconnection of S-nutrition with nitrate assimilation and that OAS plays a major role in this regulation. The study must be helpful in developing a nutrient-management technology for optimization of crop productivity.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation

In wild-type Nicotiana plumbaginifolia and other higher plants, nitrate reductase (NR) is rapidly inactivated/activated in response to dark/light transitions. Inactivation of NR is believed to be caused by phosphorylation at a special conserved regulatory Ser residue, Ser 521, and interactions with divalent cations and inhibitory 14-3-3 proteins. A transgenic N. plumbaginifolia line (S(521)) was constructed where the Ser 521 had been changed by site-directed mutagenesis into Asp. This mutation resulted in complete abolishment of inactivation in response to light/dark transitions or other treatments known to inactivate NR. During prolonged darkness, NR in wild-type plants is in the inactivated form, whereas NR in the S(521) line is always in the active form. Differences in degradation rate between NR from S(521) and lines with non-mutated NR were not found. Kinetic constants like Km values for NADH and NO3(-) were not changed, but a slightly different pH profile was observed for mutated NR as opposed to non-mutated NR. Under optimal growth conditions, the phenotype of the S(521) plants was not different from the wild type (WT). However, when plants were irrigated with high nitrate concentration, 150 mM, the transgenic plants accumulated nitrite in darkness, and young leaves showed chlorosis.     

Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools

The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.     

Differential regulation of nitrate reductase induction in roots and shoots of cotton plants

The induction of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.) was regulated by several amino acids and by ammonium. Glycine, glutamine, and asparagine strongly inhibited induction of activity by nitrate and also decreased growth of sterile-cultured roots on a nitrate medium. Methionine, serine, and alanine weakly inhibited induction, and 11 other amino acids had little or no effect. Ammonium also decreased induction in root tips, but was most effective only at pH 7 or higher. The optimum conditions for ammonium regulation of induction were identical to those for growth of sterile-cultured roots on ammonium as the sole nitrogen source. Aspartate and glutamate strongly stimulated induction, but several lines of evidence indicated that the mechanism of this response was different from that elicited by the other amino acids. The effects of amino acids on induction appeared to be independent of nitrate uptake.