Nitrate Reductase In Higher Plants Research Articles

A Model for the Circadian Oscillations in Expression and Activity of Nitrate Reductase in Higher Plants

Nitrate is the major nitrogen source for many plants. The first step of the nitrate assimilation pathway is the reduction of nitrate to nitrite, catalysed by nitrate reductase (NR). Circadian oscillations in expression and activity of NR have been demonstrated in many plant species. The pathway by which this circadian behaviour is regulated remains to be elucidated. In this study, based on recent experimental observations, a mathematical model is proposed to explain the origin of diurnal and circadian oscillations in NR gene expression and enzyme activity. The dynamic model is based on the feedback interconnections between NR and its substrate, nitrate. In the model, NR activity is regulated at the transcriptional level, in response to the balance between nitrate influx and reduction, and at the post-translational levels in response to signals from carbon assimilation. Conditions for the model system to generate self-sustained circadian oscillations are investigated by numerical simulations. Under light/dark cycles, the simulation results are in agreement with the observed diurnal pattern of changes in leaf nitrate concentration, NR transcript level and NR activity. Within a range of kinetic parameter values, circadian oscillation behaviour persists even under constant light, with periods of approx. 24 h. These simulation results suggest that sustained circadian oscillations can originate from the feedback interactions between NR and its substrate, nitrate, without the need to postulate the existence of an endogenous 'circadian clock'.

Light Regulation of Nitrate Reductase in Higher Plants: Which Photoreceptors are Involved?

Abstract: The regulation by light of nitrate reductase (NR) in higher plants is very complex. NR is firstly regulated by transcriptional control. In etiolated plants, phytochrome is the main photoreceptor involved and its low fluence response (LFR) is the common response mode. The effect of the very low fluence response (VLFR) has been reported for one of the isoforms in Arabidopsis thaliana, NIA2. The existence of a far‐red light‐mediated high irradiance response (HIR) has been demonstrated in phytochrome A over‐expressing tobacco, confirming classical photophysiological experiments. For Lemna aequinoctialis, the existence of a red light‐mediated high irradiance response has been assumed. In green plants, the effect of phytochrome on the level of NR protein is very modest, and the effect of light is mediated via photosynthesis, most probably through biochemical signals. The molecular basis of this switch from the action of phytochromes in etiolated tissue to effects of photosynthesis in green tissue is not known.The influence of light on NR is also regulated by fast post‐translational modification. This mechanism is based on the phosphorylation/dephosphorylation of a serine residue in the hinge 1 region of NR and the subsequent Mg2+/polyamine‐dependent binding of the phosphorylated form to a 14‐3‐3 protein. Several kinases phosphorylate NR, and phosphatases not only act on NR but also dephosphorylate NR kinases. No influence of phytochrome is detectable. Instead, post‐translational modification depends on the effect of photosynthesis. Photosynthesis may exert its effect on this process via biochemical products of photosynthesis (glucose, sugar phosphates), via substrates (nitrate, NADH) and/or by influencing Ca2+ flux.

Phytochrome and post-translational regulation of nitrate reductase in higher plants

The possible influence of phytochrome on the activity state of nitrate reductase (NR) was investigated in etiolated plants where the expression of the NR gene is known to be under the control of phytochrome. Activity state is defined as NR activity assayed in the presence of Mg 2+ as percentage of NR activity measured in the absence of Mg 2+. This measurement is assumed to reflect non-phosphorylated NR as percentage of total NR. Beside etiolated barley and maize leaves, a photosynthetic mutant of Lemna aequinoctialis was investigated and compared with the wild type. The increase of NR activity following a red light pulse, mediated via phytochrome, was confirmed in all etiolated plant species investigated as well as in both strains of L. aequinoctialis cultivated in glucose-containing medium. The effect of continuous red light surpassed the effect of a single red light pulse in each case. However, the results did not show any stimulating effect of phytochrome on the activity state caused by post-translational modulation. The activity state was strongly increased by continuous red light in the wild type of L. aequinoctialis but not in the photosynthetic mutant. These results show that the phytochrome system is not important for the post-translational regulation of NR.

Two cDNAs representing alleles of the nitrate reductase gene of potato (Solanum tuberosum L. cv. Desirée): sequence analysis, genomic organization and expression.

Two, different, full-length cDNAs, StNR2 and StNR3, of 3049 and 3066 nucleotides, respectively, were isolated from a Solanum tuberosum L. cv. Desirée leaf cDNA library by an RT-PCR approach. Conceptual translation of the longest open reading frame of each cDNA showed that they could encode a protein of 911 amino acids in each case with an M(r) of 102.6 and 102.5 kDa, respectively, and with 99.7% identity with each other. The cDNAs have a high degree of sequence similarity with all higher plant nitrate reductases (NRs) and possess structural characteristics expected of NADH-NR proteins, consistent with enzyme activity data. Southern analysis of genomic DNA suggested the presence of a single NR gene in the potato genome whilst studies using the mapping population F1840, and the full-length StNR2 cDNA as hybridization probe, identified a single NR locus within the potato genome that is located on chromosome XI. The two cDNAs identified here are probably derived from two transcribing alleles of this single gene. Distribution of total NR transcript and of NADH-NR activity, in different organs of compost-grown plants, depended on the level of nitrate supplied: at low nitrate level transcript and activity were detected only in leaf and stem tissue whilst at high nitrate level they could also be detected in root and stolon. An RT-PCR approach was used to differentiate between the transcripts derived from the two identified alleles and to show that the pattern of transcription of the two identified alleles of the potato nia gene, in the organs studied, is the same.

Nitrate reductase in higher plants: A case study for transduction of environmental stimuli into control of catalytic activity

In higher plants, cytosolic NAD(P)H‐nitrate reductase (NR) is rapidly modulated by environmental conditions such as light, CO2, or oxygen availability. In leaves, NR is activated by photosynthesis, reaching an activation state of 60–80%. In the dark, or after stomatal closure, leaf NR is inactivated down to 20 or 40% of its maximum activity. In roots, hypoxia or anoxia activate NR, whereas high oxygen supply inactivates NR. Spinach leaf NR is inactivated by phosphorylation of serine 543 and subsequent Mg2+‐dependent binding of 14‐3‐3 proteins at, or close to, this phosphorylation site. At least three different protein kinases (NR‐PK) have been identified in spinach leaves that are able to phosphorylate NR on serine 543. Two of them show up as calmodulin‐like domain protein kinases (CDPKs), and one as a SNF1‐like protein kinase. Dephosphorylation of serine 543 is catalyzed by a Mg2+‐dependent protein phosphatase and by a type 2A protein phosphatase (NR‐PP), which is regulated by a trimer/dimer interconversion. The NR‐PKs, NR‐PPs, and 14‐3‐3s are present even in NR‐depleted plant tissues. Artificial activation of NR in vivo is achieved by cellular acidification, by respiratory inhibitors, or by mannose feeding. As for anoxia, these treatments seem to act, at least in part, via cytosolic acidification, mediated by low cytosolic ATP levels. Activation is also achieved by ionophore‐induced release of divalent cations from the cytosol. In addition, cytosolic AMP and phosphate esters seem to regulate NR‐PK and NR‐PP activities, thereby adapting NR activity within minutes to the changing environment.

A conserved acidic motif in the N-terminal domain of nitrate reductase is necessary for the inactivation of the enzyme in the dark by phosphorylation and 14-3-3 binding.

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611-621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.

Identification of an endogenous NADPH-regenerating system coupled to nitrate reduction in vitro in plant and fungal crude extracts

A low molecular weight factor obtained from the crude extract of nit-1 mutant of Neurospora crassa (NEF) was capable of stimulating the activity of reconstituted NADPH specific nitrate reductase (NR, EC 1.6.6.3; Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A low molecular weight regulator of nitrate reductase in higher plants. 4th International Symposium on Inorganic Nitrogen Assimilation and 1st Fohs Biostress Symposium. Seeheim/Darmstadt, Germany, 1995, p. 9). This factor eluted from a G15 column as glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P). Like the hexose-phosphates, NEF allowed rapid regeneration of NADPH from the NADP +, resulting from nitrate reduction. Sequential treatment of the NEF-containing fraction with commercial preparations of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.43) and phosphoglucose isomerase (PGI, EC 5.3.1.9) resulted in the oxidation of NEF and the reduction of NADP +. NEF, therefore, was a substrate of G6PDH and 6PGDH in a reaction which reduced NADP + to NADPH. It was concluded that the low molecular weight factor enhancing NR activity isolated from fungal and plant crude extracts (Z.Z. Alikulov, N.A. Savidov, H.S. Lips, A low molecular weight regulator of nitrate reductase in higher plants. 4th International Symposium on Inorganic Nitrogen Assimilation and 1st Fohs Biostress Symposium. Seeheim/Darmstadt, Germany, 1995, p. 9) may be a mixture of hexose phosphates. G6PDH, 6PGDH and PGI were active in crude extracts of plants and fungi used for NR assays. Oxidation of one mole of G6P led to the production of approximately two moles of NADPH. The activities of PGI, G6PDH and 6PGDH exceeded that of NR in both fungal and plant crude extract. Fungal crude extracts exhibited a considerably higher capacity to regenerate NADPH than plant extracts. The addition of G6P during the in vitro NR assay increased the stability of the reaction allowing detection of very low NR activity levels. The stimulation of NR in vitro by NADPH generating systems points out the physiological importance of these systems in nitrate assimilation and in the nitrate reduction assays in situ and in vivo.

Light-mediated Regulation of Nitrate Reductase in Higher plants

Regulation of nitrate reductase (NR) by light is a complex process and is manifested through gene expression, both at the level of transcription and translation, covalent modification of the enzyme and supply of reductant. Light induces nia gene transcription by some still unknown mechanism. Light induction of nia mRNA is also mediated via phytochrome. By using protein synthesis inhibitors it has been observed that light enhances translation of NR mRNA. Light induction of NR synthesis follows the circadian rhythmicity. Cytokinin increases NR activity by regulating NR expression at the transcriptional level. In response to light-dark transition, NR is quickly inactivated/activated by phosphorylation/dephosphorylation. Inactivation of NR involves phosphorylation of the serine-543, located at the hinge 1 region connecting MoCo domain and cyt b domain of NR, by a Mg2+ dependent protein kinase and subsequent binding of an inhibitor protein. A type 2A protein phosphatase dephosphorylates/activates NR in response to light signal. Light also regulates supply of reductant via photosynthesis for activity of NR. Whether light is absolutely necessary for NR activity is also discussed.

Post-transcriptional regulation of nitrate reductase by light is abolished by an N-terminal deletion.

Higher plant nitrate reductases (NRs) carry an N-terminal domain whose sequence is not conserved in NRs from other organisms. A gene composed of a full-length tobacco NR cDNA with an internal deletion of 168 bp in the 5' end fused to the cauliflower mosaic virus 35S promoter and appropriate termination signals was constructed and designated as delta NR. An NR-deficient mutant of Nicotiana plumbaginifolia was transformed with this delta NR gene. In transgenic plants expressing this construct, NR activity was restored and normal growth resulted. Apart from a higher thermosensitivity, no appreciable modification of the kinetic parameters of the enzyme was detectable. The post-transcriptional regulation of NR by light was abolished in delta NR transformants. Consequently, deregulated production of glutamine and asparagine was detected in delta NR transformants. The absence of in vitro delta NR activity modulation by ATP suggests the impairment of delta NR phosphorylation and thereby suppression of delta NR post-translational regulation. These data imply that post-transcriptional control of NR expression is important for the flow of the nitrate assimilatory pathway.

Posttranslational Regulation of Nitrate Reductase in Higher Plants.

In many plants, nitrogen represents 2 to 6% of dry matter, most of it being present in the form of amino acids, proteins, or nucleic acids. In these organic compounds, nitrogen exists in its most reduced state (oxidation state -3). It is taken up from the soil primarily in the form of nitrate (oxidation state +5). Thus, nitrate has to be reduced by plants at the expense of reductants such as NADH or NADPH, requiring 8 mol of electrons [or 4 mol of NAD(P)H] per mol of nitrate. Reduction is a two-step mechanism. The first step, reduction of nitrate to nitrite (+3 oxidation state), is catalyzed by assimilatory NR, which is an NAD(P)H-dependent cytosolic enzyme. Nitrite is further reduced to ammonia (-3 oxidation state) in the plastids of leaves or roots by NiR. There are at least two important reasons why plants must exert control on the velocity of nitrate reduction. First, it is an energy-consuming process. As shown above, 8 electrons are required to reduce one nitrate to ammonium, but only 4 electrons are needed to reduce CO2 to the carbohydrate level. Accordingly, a C/N ratio of 10 in the plant biomass (a value found in many herbaceous plants) indicates that about 20% of the photosynthetically produced electrons are consumed for nitrate reduction. Plants have to avoid 'luxury' consumption of nitrate and energy. Second, and perhaps more important, the primary product of nitrate reduction, nitrite (NO2-), is cytotoxic and regarded to be mutagenic as a result of the ability to diazotize amino groups. HNO2 is also a weak acid that, in its undissociated form, can easily penetrate biomembranes, thereby leading to acidification of cells or subcellular compartments. Thus, it makes sense if nitrate reduction is controlled in such a way that it does not exceed nitrite and ammonium consumption. In fact, even under rapidly fluctuating environmental conditions, nitrite levels in plant tissues remain low (below 0.1 mM). Apparently, the overall rates of nitrate and nitrite reduction always match the availability of energy and of carbohydrate, perhaps with the exception of some extreme conditions to be discussed below. Synchronization of nitrate reduction and carbon metabolism may occur at the level of transcription or translation of participating enzymes. The expression of NR genes at the transcription level is highly affected by nitrate, but also by light, plant hormones, and other factors (for review, see Solomonson and Barber, 1990; Lillo, 1994). The enzyme protein is rather short-lived, being degraded with a half-time of a few hours (Li and Oaks, 1993). This high turnover rate permits control of nitrate reduction, e.g. in response to nitrate availability (Li and Oaks, 1993). However, over the years, a number of situations have been described in which the level of extractable NRA did not match NR protein or the rate of nitrate reduction in vivo, indicating that yet other regulatory mechanisms might exist that modulate the catalytic activity of the protein (Lillo, 1994, and refs. cited therein). Such a newly discovered type of NR modulation, a reversible protein phosphorylation, will be described below.

Light/dark regulation of higher plant nitrate reductase related to hysteresis and calcium/magnesium inhibition

Light/dark regulation of higher plant nitrate reductase related to hysteresis and calcium/magnesium inhibition

Structure, function and regulation of nitrate reductase in higher plants

Structure, function and regulation of nitrate reductase in higher plants

Structure, function and regulation of nitrate reductase in higher plants

Two types of nitrate reductase (NR) have been recognized in higher plants. Most plants contain a NADH‐specific NR, a few plants contain only a NAD(P)H‐bispecific NR and some plants contain both NR types. Most NRs are nitrate inducible. Many NRs are expressed in both roots and leaves, but in some species a root‐specific NR has been identified. All NRs from higher plants are dimers of identical subunits with 881 (bean) to 926 (spinach) amino acid residues. The complete nucleotide sequence of NR genes or cDNAs from several higher plants have been obtained. The amino acid identity between the species analyzed ranges from 60 to 90%. NR is organized into 3 domains containing FAD, heme and a molybdenum cofactor. Electrons are transferred from NAD(P)H to nitrate via these cofactors. Nitrate triggers transcription of inducible genes for NR. NR activity and mRNA accumulation can be induced to a low level in etiolated and green dark‐adapted plants by addition of nitrate. The accumulation of NR mRNA and activity is strongly stimulated by subsequent exposure to white light. Light also stimulates NR mRNA translation and/or the stability of the NR protein. In light/dark grow plants the mRNA level generally peaks at the end of the dark periods and the NR activity peaks a few hours later. These diurnal oscillations are controlled via transcription initiation.

The Aspergillus niger niaD gene encoding nitrate reductase: upstream nucleotide and amino acid sequence comparisons

The Aspergillus niger niaD gene has been sequenced and the inferred nitrate reductase (NR) protein found to consist of 867 amino acid residues (97 kDa). The gene is interrupted by six small introns, as deduced by comparison with the niaD gene of Aspergillus nidulans. The positions of these putative introns are conserved between the two fungi, although the sequences are dissimilar. The niiA gene, encoding nitrite reductase, the second reductive step in the nitrate assimilation pathway, is tightly linked to niaD and divergently transcribed in A. niger, similar to the general organisation in the related fungi, Aspergillus oryzae and A. nidulans. The nucleotide (nt) sequences of the intergenic region between niiA and niaD (excluding the ATG translation start codon) of A. niger (1668 nt) and A. oryzae (1575 nt) were determined and compared with the previously determined A. nidulans (1262 nt) sequence. No striking extended nt regions of homology are observed in spite of the fact that transgenic strains with fungal niaD or the two control genes required for induction and repression show virtually normal regulation. Fungal NR shows considerable aa homology with higher plant NR, particularly within the co-factor domains for flavin adenoside dinucleotide, heme and molybdopterin cofactor.

Sequence of a cDNA encoding the bi-specific NAD(P)H-nitrate reductase from the treeBetula pendula and identification of conserved protein regions

Nitrate reductase (NR) assays revealed a bispecific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)+ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises a reading frame encoding a protein of 898 amino acids which reveals 67%-77% identity with NADH-nitrate reductase sequences from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates that the bi-specific NR is encoded by a single copy gene in birch.

High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B 5 reductase

Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin. The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced. A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c. When this construct was expressed in E. coli, a 30 kD polypeptide was found to be newly synthesized. The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD. The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 μmol NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b 5 reductase.

Immunological Comparisons of Nitrate Reductase of Different Plant Species Using Monoclonal Antibodies

Six monoclonal antibodies against different epitopes of maize leaf nitrate reductase were used to compare plant nitrate reductases in enzyme linked immunosorbent assay and enzyme activity inhibition tests. The number of cross-reacting antibodies was shown to vary with species according to phylogenetic classification, ranging from five (sugarcane) to one (dicotyledonous species). Cross-reactions were restricted to higher plant nitrate reductases.

Reversible inactivation of wheat leaf nitrate reductase by NADH, involving superoxide ions generated by the oxidation of thiols and FAD

The conversion of wheat leaf NADH-nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) to a reduced inactive form, by preincubation with NADH (in the absence of nitrate), occurred in the presence of either dithiothreitol and/or FAD but not with cysteine. This inactivation of nitrate reductase, unlike that by cyanide, was dependent on aerobic conditions and was prevented by EDTA and superoxide dismutase. Superoxide ions were produced by the auto-oxidation of dithiothreitol but not cysteine, at pH 7.5. Thus, superoxide ions can mediate the inactivation of NADH-reduced nitrate reductase in higher plants. Pretreatment of nitrate reductase with NADH alone (over-reduction) did not inactivate the enzyme. A nucleophilic agent, i.e., cyanide or superoxide is necessary to inhibit electron transfer by the enzyme to nitrate. Nitrite or azide were not effective. Data in the literature which suggest that NADH stabilizes nitrate reductase, rather than resulting in its inactivation, can now be explained. In these cases, cysteine was used as thiol and FAD was not added in the extraction or incubation media so that no superoxide ion was produced.

Monoclonal antibodies to a higher-plant nitrate reductase: Differential inhibition of enzyme activities

A set of monoclonal antibodies has been raised against NADH-nitrate reductase (NR; EC 1.6.6.1) from spinach (Spinacea oleracea L.) leaves. Antibodies were screened by enzyme-linked immunosorbent assay and by their ability to inhibit various activities of the enzyme. The six monoclonals selected (AFRC MAC 74 to 79) are all gamma globulins; four (MAC 74 to 77) inhibit all terminal donating activities (NADH-NR; flavin mononucleotide, reduced form (FMNH2)-NR; and methyl viologen, reduced form (MV)-NR) and two (MAC 78 and 79) inhibit the acceptor activities (NADH-NR, and NADH-cytochrome c reductase). MAC 74 to 77 inhibit the NADH-NR activity of crude extracts of a variety of species (mono- and dicotyledoneae) while MAC 78 and 79 are effective against spinach and marrow, but not oil-seed rape, cucumber, oats, wheat and barley.

Influence of Incubation pH on in vivo Nitrate Reductase Activity in Leaves of Two Perennial and Two Annual Tropical Plants

Summary The effect of assay medium pH on in vivo nitrate reductase (NR) activity of cashew ( Anacardium occidentale L.) leaves was studied. The enzyme activity continued to increase beyond pH 7.5 up to pH 10 until the leaf discs showed symptoms of damage. Entry of nitrate into the leaf tissue also increased up to pH 9. Higher activities of NR at alkaline pH were also observed in cacao, country bean ( Dolichos lablab ) and sugarcane leaves. This is perhaps the first time that such a high range is reported as optimal pH for NR in higher plants.