Nitrate Reductase Protein Research Articles

Modulation of carbon and nitrogen metabolism, and of nitrate reductase, in untransformed and transformed Nicotiana plumbaginifolia during CO 2 enrichment of plants grown in pots and in hydroponic culture

Transformed plants of Nicotiana plumbaginifolia Viv. constitutively expressing nitrate reductase (35S-NR) or β-glucuronidase (35S-GUS) and untransformed controls were grown for two weeks in a CO2-enriched atmosphere. Whereas CO2 enrichment (1000 μl · l−1) resulted in an increase in the carbon (C) to nitrogen (N) ratio of both the tobacco lines grown in pots with vermiculite, the C/N ratio was only slightly modified when plants were grown in hydroponic culture in high CO2 compared to those grown in air. Constitutive nitrate reductase (NR) expression per se did not change the C/N ratio of the shoots or roots. Biomass accumulation was similar in both types of plant when hydroponic or pot-grown material, grown in air or high CO2, were compared. Shoot dry matter accumulation was primarily related to the presence of stored carbohydrate (starch and sucrose) in the leaves. In the pot-grown tobacco, growth at elevated CO2 levels caused a concomitant decrease in the N content of the leaves involving losses in NO− 3 and amino acid levels. In contrast, the N content and composition were similar in all plants grown in hydroponic culture. The 35S-NR plants grown in air had higher foliar maximum extractable NR activities and increased glutamine levels (on a chlorophyll or protein basis) than the untransformed controls. These increases were maintained following CO2 enrichment when the plants were grown in hydroponic culture, suggesting that an increased flux through nitrogen assimilation was possible in the 35S-NR plants. Under CO2 enrichment the NR activation state in the leaves was similar in all plants. When the 35S-NR plants were grown in pots, however, foliar NR activity and glutamine content fell in the 35S-NR transformants to levels similar to those of the untransformed controls. The differences in NR activity between untransformed and 35S-NR leaves were much less pronounced in the hydroponic than in the pot-grown material but the difference in total extractable NR activity was more marked following CO2 enrichment. Foliar NR message levels were decreased by CO2 enrichment in all growth conditions but this was much more pronounced in pot-grown material than in that grown hydroponically. Since β-glucuronidase (GUS) activity and message levels in 35S-GUS plants grown under the same conditions of CO2 enrichment (to test the effects of CO2 enrichment on the activity of the 35S promoter) were found to be constant, we conclude that NR message turnover was specifically accelerated in the 35S-NR plants as well as in the untransformed controls as a result of CO2 enrichment. The molecular and metabolic signals involved in increased NR message and protein turnover are not known but possible effectors include NO3 −, glutamine and asparagine. We conclude that plants grown in hydroponic culture have greater access to N than those grown in pots. Regardless of the culture method, CO2 enrichment has a direct effect on NR mRNA stability.

Correlation between apparent activation state of nitrate reductase (NR), NR hysteresis and degradation of NR protein

Nitrate reductase (NR) activity was measured in extracts from spinach leaves exposed to light or prolonged darkness, and to various treatments provoking an artificial activation of the enzyme in the dark. NR activity was determined immediately either in the presence of Mg2+, which gives an estimation of the putative (actual) activity in situ (NRact), or in EDTA without preincubation, which gives an intermediate activity (NRint), or after a 30 min preincubation with EDTA plus AMP plus Pi, which gives the maximum NR activity (NRmax). NRmax is thought to reflect total NR protein contents. In the dark, NRact was usually very low. Dark inactivation was prevented or reversed by feeding AICAR (5-aminoimidazole-4-carboxiamide ribonucleoside), or by anaerobiosis, acid treatment or addition of uncoupler. During prolonged darkness, NRmax decreased, indicating net protein degradation with a half-time of 21 h. Conditions which caused an activation (dephosphorylation) of NR in the dark, slowed down NR protein degradation. This was also confirmed by Western blotting. Blockage of cytosolic protein synthesis with cycloheximide (CHX) did not accelerate NR protein degradation. In contrast, after 5 h in the dark, NRact increased in CHX-treated leaves. As this increase was sensitive to PP2A-inhibitors, it was probably due to NR dephosphorylation. However, extractable NR kinase and NR phosphatase activities were not changed by CHX treatment. Apparently, CHX interacted with the NR regulatory system indirectly by affecting turnover of another protein. The increase from NRint to NRmax which occurred during preincubation of the leaf extract with EDTA plus AMP plus Pi was insensitive to PP2A inhibitors and was interpreted as a hysteretic conversion of NR from an inactive into an active form. Hysteretic activation was positively correlated to the NR phosphorylation state. A model is presented to explain the hysteretic behaviour of NR in relation to NA phosphorylation/ dephosphorylation. Overall, the data indicate that NR protein phosphorylation not only controls the catalytic activity of NR, but also acts as a signal for NR protein degradation, with phospho-NR probably being a better substrate for protein degradation than the dephospho-form.

Effect of cinnamate on nitrate reductase activity in isolated cucumber cotyledons

The effect of cinnamic acid on in vivo nitrate reductase activity and protein content in cucumber cotyledons was studied. Cinnamate increased in vivo nitrate reductase activity and also the total protein content at lower concentrations (0.01–0.1 mM). Higher concentration, however, proved inhibitory. The effect of cinnamate on nitrate reductase activity has been discussed.

Nitrate reductase expression in maize leaves (Zea mays) during dark‐light transitions. Complex effects of protein phosphatase inhibitors on enzyme activity, protein synthesis and transcript levels

The effects of cytoplasmic protein synthesis and protein phosphatase activity on NADH:nitrate reductase (NR) activity, protein and transcript were examined in maize (Zea mays L.) seedling leaves. A rapid increase in NR activity, measured in the presence of 5 mM Mg2+, was found upon exposure of excised leaves to light. Inhibitors of protein phosphatase activity (okadaic acid [OKA] and microcystin [MC]‐LR) completely prevented the increase in NR activity. The cytoplasmic protein synthesis inhibitor, cycloheximide (CHX), did not affect Mg2+ inhibition of NR activity during the dark‐to‐light transition. Vmax NR activity, measured in the presence of 5mM Mg2+, was found upon exposure of excised leaves to light. Inhibitors of protein phosphatase activity (okadaic acid [OKA] and microcystin [MC]‐LR) completely prevented the increase in NR activity. The cytoplasmic protein synthesis inhibitor, cycloheximide (CHX), did not affect Mg2+ inhibition of NR activity during the dark‐to‐light transition. Vmax NR activity, measured in the presence of Pi and EDTA, remained constant or increased slightly in maize leaves during the first 2 h of the light period. OKA, MC‐LR or CHX treatment caused a 40 to 50% reduction in Vmax NR activity during this time. Incorporation of 35S‐Met into NR protein was reduced more than 90% by CHX and 80% by OKA. The inhibition of NR protein synthesis by CHX and OKA correlated with a 50 to 60% decrease in 35S‐Met incorporation into total soluble protein over the treatment period. The increase in NR mRNA levels early in the light period was prevented by OKA and MC‐LR, but not by CHX. OKA had a similar effect on sucrose phosphate synthase mRNA levels, but did not affect Catalasel or Catalase3 mRNA accumulation. The data suggest that light‐induced decreases in Mg2+ inhibition of NR activity and transcript levels are independent of new protein synthesis. The effects of OKA and MC‐LR indicate that protein phosphatase activities could be involved, directly or indirectly, in the regulation of NR activity, protein synthesis and transcript accumulation.

Phosphorylation/dephosphorylation steps are key events in the phytochrome-mediated enhancement of nitrate reductase mRNA levels and enzyme activity in maize.

We provide evidence to show that the increase in nitrate reductase (NR) transcript level stimulated by red light is mediated via a phosphorylation-dependent step. The light-stimulated enhancement of NR transcript level was significantly inhibited by H-7, a protein kinase inhibitor, whereas okadaic acid (OKA), a phosphatase inhibitor, had no effect. Phorbol myristate acetate (PMA), an activator of protein kinase C (PKC) enhanced the NR transcript level in dark-grown leaves. No correlation between changes in NR transcript level and NR activity (NRA) was observed. Inhibition of NRA by OKA and stimulation by H-7 indicated that NRA is increased by dephosphorylating the enzyme. We have identified a protein kinase (C type) that can phosphorylate the purified NR in vitro without the involvement of other accessory proteins. By in vivo labelling with 32P and immunoprecipitation of NR with NR antibodies it was found that in the presence of OKA most NR protein (NRP) was present in phosphorylated state, while with H-7 the reverse was seen. The red (R) and far-red (FR) light reversible experiments suggested that phytochrome (Pfr, an active form) stimulation of NRA is mediated by dephosphorylation of the enzyme, suggesting that Pfr regulates both NR transcription and NRA via phosphorylation/dephosphorylation steps controlled by separate signal transduction pathways.

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

Maize (Zea mays L.) grown on low (0.8 mM) NO 3 - , as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO 3 - on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg2+. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO 3 - did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO 3 - content and the NR activation state. Similarly, the NR activation state did not respond to NO 3 - . Since the NR activation state determined from the degree of Mg2+-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO 3 - -induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO 3 - . We conclude that NO 3 - does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C3 species) or maize (a C4 species). The short-term regulation of NR therefore differs from the NO 3 - -mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.

Effects of NO (+ NO2) pollution on growth, nitrate reductase activities and associated protein contents in glasshouse lettuce grown hydroponically in winter with CO2 enrichment

summaryWinter hydroponic growth of several lettuce cultivars under glass showed considerable inhibition (up to 47%) of growth after 6 wk exposure to concentrations of NO (+ NO2 450 nl −1 in total) typical of emissions from propane burners used for direct heating and CO2 enrichment. After a further 4 wk under similar conditions, however, these growth depressions were replaced by a swing into benefit so that, by harvest, pollutant‐exposed lettuces were bigger and had faster assimilation rates than those growing in clean CO2‐enriched air. This adaptation may partly be explained by enhanced use of NO2‐derived N by lettuce leaves, a consequence of increased nitrate reductase (NaR) activities and amounts of associated NaR proteins, despite adequate nitrate also being available in the hydroponic fluid. Rates of NaR activity in the roots, by contrast, were depressed by NO (+ NO2) pollution. NaR activities were highest in early afternoon in clean or polluted air but these daily patterns did not coincide with the content of NaR‐associated proteins determined by ELISA. Other mechanisms of modulating NaR activity must therefore be responsible.

The protein kinase, protein phosphatase and inhibitor protein of nitrate reductase are ubiquitous in higher plants and independent of nitrate reductase expression and turnover

Nitrate reductase activity and NR protein levels in various leaf tissues were drastically decreased (<3.5% of normal activity) either by keeping detached leaves in continuous darkness for up to 6 d (spinach), or by growing plants (pea, squash) hydroponically on ammonium as the sole N-source, or by germinating and growing etiolated seedlings in complete darkness (squash). The presence of nitrate reductase protein kinase (NRPK), nitrate reductase protein phosphatase (NRPP) and inhibitor protein (IP) was examined by measuring the ability of NR-free desalted extracts to inactivate (ATP-dependent) and reactivate (5′-AMP/EDTA-dependent) added purified spinach NR in vitro. Extracts from low-NR plants (ammonium-grown pea and squash) were also prepared from leaves harvested at the end of a normal light or dark phase, or after treating leaves with anaerobiosis, uncouplers or mannose, conditions which usually activate NR in nitrategrown normal plants. Without exception, extracts from NR-deficient plant tissues were able to inactivate and reactivate purified spinach NR with normal velocity, irrespective of pretreatment or time of harvest. Considerable NRPK, NRPP and IP activities were also found in extracts from almost NR-free ripe fruits (cucumber and tomato). Activities were totally absent, however, in extracts from isolated spinach chloroplasts. The NRPK and IP fractions were partially purified with normal yields from NR-deficient squash or spinach leaves, following the purification protocol worked out for nitrate-grown spinach. The Ca2+/Mg2+-dependent kinase fraction from NR-deficient squash or spinach phosphorylated added purified spinach NR with γ-[32P]ATP and inactivated the enzyme after addition of IP. It is suggested (i) that the auxiliary proteins (NRPK, IP, NRPP) which modulate NR are rather species- or organ-unspecific, (ii) that they do not turn over as rapidly as does NR, (iii) that they are probably expressed independently of NR, and (iiii) that they are not covalently modulated, but under control of metabolic and/or physical signals which are removed by desalting.

Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase.

Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.

Changes in nitrate reductase activity and the protective effect of molybdenum during cold stress in winter wheat grown on acid soil

Summary Two cultivars (frost resistant Sadovo 1 and frost susceptible San Pastore) of winter wheat (Triticum aestivum L.) were used to study the protective effect of molybdenum during freezing of plants grown on acid soil. We followed the effect of cold acclimation (2°C for 7 days) and freezing (−5 °C for 16 h) on the activities of nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) in the leaves of both wheat cultivars grown on soil with pH 6.0 and pH 4.5. No difference was observed between the cultivars in the temperature induced changes of enzyme activities. During cold acclimation the activities of the three enzymes increased irrespective of the soil's pH; this increase, which was most pronounced for NADH: NR activity, was paralleled by an increase in NR protein. Freezing affected only NR activity in plants grown on acid soil; the sharpest decline was found for NADH : NR activity and FADH : NR partial activity. Molybdenum supplied to winter wheat cv. Sadovo 1 grown on soil with pH 4.5 prevented the frost-induced decline of NADH : NR activity. The possible mechanism of the protective effect is discussed.

Xylem exudation is related to nitrate assimilation pathway in detopped maize seedlings: use of nitrate reductase and glutamine synthetase inhibitors as tools

The xylem exudation of detopped 7-d-old seedlings of Zea mays L. doubled when KCl was present in the root medium compared to seedlings maintained on water. It was further enhanced when KCl was replaced by nitrogen compounds such as nitrate, ammonium and glutamine. The role of the nitrate assimilation pathway on the enhancement of xylem exudation rate was investigated using tungstate, an inhibitor of nitrate reductase (NR) activity, and phosphinothricin or methionine sulphoximine, inhibitors of glutamine synthetase (GS) activity. The sap levels of NO 3 - , NH 4 + , glutamine, and asparagine was used to ascertain the in vivo inhibition of both enzymes. The tungstate effects were also checked by measuring leaf in vitro NR activity and NR protein content. Xylem exudation rate of detopped seedlings fed with KNO 3 decreased when the nitrate assimilation pathway was blocked either at the NR or at GS sites. This decrease was prevented when urea (acting as NH 4 + supply) was given simultaneously with tungstate. KNO 3 does not act directly on exudation, but through the involvement of NH 4 + . The involvement of glutamine was also shown since GS inhibition resulted in a cancellation of the enhancing effect of KNO 3 on exudation. As change of exudation rate was not linked to change in sap osmolarity, it is assumed that the assimilation chain could modify root water conductance. The role of glutamine was discussed.

Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO 3 − content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO 3 − content was observed in plants grown in nitrogen replete conditions at low irradiance (170 μmol photons·m−2 ·s−1). This relationship disappeared at higher irradiance (450 μmol photons·m−2·S−1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO 3 − accumulated in the leaves of plants grown at 450 μmol photons·m−2·s−1 than in those grown at 170 μmol photons·m−2·s−1 in N-replete conditions. The foliar NO 3 − level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO 3 − was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO 3 − in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.

Regulation of Nitrate Reductase during Early Seedling Growth (A Role for Asparagine and Glutamine).

Growth systems that either permit (wet system) or prevent (dry system) the hydrolysis of endosperm reserves in maize (Zea mays) seedlings were developed to study the effect of endosperm reserves on the acquisition of external nitrogen. Three-day-old seedlings treated with 5 mM KNO3 for 24 h had higher levels of nitrate reductase (NR) activity and protein in shoot and root tissues in the dry relative to the wet system. This suggests that the induction of NR is sensitive to products of hydrolysis of endosperm reserves. Asparagine (1 mM) or glutamine (1 mM), potential products of that hydrolysis, inhibited the induction of NADH-dependent root NR in the dry system by about 70%. The inhibition of the induction of NR activity in the wet system was only about 35%, suggesting that the enzyme in the wet system was already partially repressed at 3 d. At 5 d, when asparagine and glutamine levels in the plant tissue had decreased, the induction of root NR activity was inhibited to a similar extent in the two growth systems by amide additions. The shoot enzyme was less sensitive to amide additions, and 10 mM concentrations of either amide was required for a 65% inhibition.

Identification and characterization of a nitrate reductase gene from bean (Phaseolus vulgaris) containing four introns

A structural gene encoding nitrate reductase (NR) in bean (Phaseolus vulgaris) has been cloned and sequenced. The NR gene encodes a protein of 890 amino acids with a molecular mass of 100 kDa. Comparison to the other known NR gene from bean reveals 76% amino acid identity and comparison to NRs from other species shows amino acid identities ranging from 67 to 77%. At three positions the amino acid sequence displays differences from residues conserved in all other known NR proteins. The coding sequence is interrupted by four introns. Three of them are located at conserved positions in the region encoding the molybdenum cofactor‐binding domain. The fourth intron is located in the hinge region between the heme and the FAD domain. This is the only example in which more than three introns have been found in a higher plant NR gene. The mRNA cap site was identified as an adenosine 79 nucleotides (nt) upstream of the ATG translation start codon. Northern analysis shows that the gene is nitrate inducible and highly expressed in trifoliolate leaves of 20‐day‐old bean plants and only weakly expressed in roots. The gene is also induced by light and sucrose in leaves of dark‐adapted plants. The mRNA displays diurnal oscillation under the control of a circadian rhythm. Putative conserved GATA motifs in the promoter are discussed.

Effect of Mg, Zn and Mo salts on nitrate reductase activity and soluble protein content in leaves ofQuercus serrata

Leaves ofQuercus serrata seedlings were sprayed with solutions of different concentrations of Mg, Zn and Mo-salts and activity of nitrate reductase (NRA) and soluble protein contents were determined in fresh leaves of different ages. The optimum dose for magnesium sulphate and zinc sulphate was 2.5 mM while for ammonium molybdate it was 0.01 mM. Under optimum doses of these nutrients high levels of NRA and protein contents were maintained upto 28 d of spray. Observations on NRA were further confirmed by leaf direct treatment and excised shoot-dipping experiments which showed maximum NRA at comparatively much lower concentrations of ions than those used for spray.

Induction and Turnover of Nitrate Reductase in Zea mays (Influence of Light).

Both light and NO3- are necessary for the appearance of nitrate reductase (NR) activity (NRA) in photosynthetic tissues. To define the light effect more precisely, we examined the response to light/dark transitions on NRA, NR protein (NRP), and NR mRNA in 6-d-old maize (Zea mays cv W64A x W182E) seedlings that had been grown in a light/dark regime for 5 d and then induced with 5 mM KNO3 for 24 h. The decay of NRA and NR mRNA in the shoot was immediate, but there were only minor changes in NRP during the initial 4 h in the dark. In root tissues, in contrast, there was a 4-h delay in the loss of NRA, NRP, and NR mRNA after transfer to the dark. When the seedlings were returned to light after a 2-h interval in the dark, shoot NRA reached 92% of the initial levels within 30 min of illumination. These results indicate that in the shoots (a) NR message production requires light and (b) the NRP that appears with light treatment and that is active is inactivated in the dark. The NRP can be reactivated when the light is turned on after short periods of darkness (2 h). Root tissues, on the other hand, probably respond to the supply of photosynthetically produced metabolites rather than to immediate products of the light reactions of photosynthesis.

Isolation and Characterization of Nitrate Reductase-Deficient Mutants of Cultured Spinach Cells: Biochemical, Immunological and mRNA Analysis

Nitrate reductase (NR)-deficient mutants of cultured spinach cells were selected by chlorate resistance. Two mutants (12F and I-1 cell lines) were isolated and analyzed for activities, protein levels, and the expression of mRNAs of NR and nitrite reductase (NiR). When mutant cells were transferred to a medium containing 20 mM KNO 3 and 20 mM NH 4 NO 3 as a nitrogen source, the 12F cell line had no detectable NR activity or NR protein, but the NR mRNA was expressed clearly in contrast with the data of activity and protein levels. The NiR activity and protein were still induced and the NiR mRNA was also expressed clearly. In the I-1 cell line, however, the NR activity and protein were still present at low levels, and the NiR activity and protein were no longer inducible. Extremely low levels of the NR and NiR mRNAs were found in the I-1 cell line. These results suggest that the 12F and I-1 cell lines are structural gene mutant and regulatory gene mutant, respectively.

Possible role for mRNA stability in the ammonium-controlled regulation of nitrate reductase expression.

Ammonium, or a metabolite of ammonium, represses the expression of nitrate reductase (NR) in Chlorella vulgaris. The removal of ammonium and addition of nitrate (induction) resulted in a rapid (20 min) peaked synthesis of NR mRNA. Nitrate reductase protein and activity increased at a much lower rate, reaching their maxima by 8 h. Ammonium added to nitrate-grown cells resulted in a dramatic decrease in NR mRNA from a steady-state level to undetectable levels within 15 min of ammonium addition. Nitrate reductase activity and protein levels decreased to 20% and 40% of initial levels respectively over 8 h. The half-life for NR mRNA under these conditions was estimated to be less than 5 min, compared with 120 min for NR protein. Such rapid decreases in NR mRNA suggested a degradation and/or cessation in NR mRNA transcription. No apparent difference in NR mRNA-specific RNAase activity of crude cell extracts (NR-induced or repressed) was observed. However, a significant difference in the susceptibility to degradation of NR mRNA from long-term nitrate-grown cells compared with the NR mRNA isolated from short-term induced cells (20 min in nitrate) was observed. NR mRNA isolated from long-term-nitrate-grown cells was completely degraded by RNAases in cell extracts under conditions in which the NR mRNA isolated from short-term induced cells was resistant to degradation. These results suggest that mRNA stability may be an important factor in the metabolic regulation of assimilatory nitrate reductase in Chlorella.

Effects of Atmospheric NO2 on Azolla-Anabaena Symbiosis

Cultures of the water fern Azolla pinnata R, Br. exposed for 1 week to atmospheric NO2 (50, 100 or 200 nl l-1) induced additional levels of nitrate reductase (NaR) protein and nitrite reductase (NiR) activity. At low concentrations of NO2 (50 nl l-1), nitrate derived from NO2 provides an alternative N source for Azolla but does not affect rates of acetylene reduction. However, the symbiotic relationship between Azolla and its endosymbiont, Anabaena azollae is only affected adversely by high concentrations (100 and 200 nl l-1) of atmospheric NO2. The resultant decreases in rate of growth, nitrogen fixation, heterocyst formation, and overall nitrogen cycling are probably due to the additional accumulation of N products derived from higher levels of atmospheric NO2. Parallel increases in levels of polyamines suggest that Azolla partially alleviates these harmful effects by incorporating some of the extra NO2-induced N into polyamines.

Evidence that the glutamine-stimulated loss of nitrate reductase protein from the yeast Candida nitratophila is not the result of inducer exclusion.

Synthesis of nitrate reductase protein and increases in nitrate reductase activity occurred in cultures of the yeast Candida nitratophila when they were incubated in medium containing ammonium nitrate. Similar treatment with glutamine plus nitrate resulted in little increase in nitrate reductase activity, in cultures grown previously with reduced nitrogen compounds, and decreases in enzyme activity, in cultures adapted to nitrate. Labelling studies conducted in vivo revealed a rapid cessation of de novo nitrate reductase synthesis when glutamine was supplied to nitrate-adapted cultures in the presence of nitrate. Intracellular glutamine concentrations increased rapidly under these conditions and these cultures exhibited high glutamine: glutamate ratios. As nitrate was taken up in the presence of glutamine in these experiments, it is concluded that the glutamine-stimulated inhibition of nitrate reductase synthesis is a consequence of repression and rapid turnover of nitrate reductase mRNA and not inducer (nitrate) exclusion.