Appearance of nitrite reductase in cotyledons of the mustard (Sinapis alba L.) seedling as affected by nitrate,phytochrome and photooxidative damage of plastids

Nitrite reductase (NIR; EC 1.7.7.1) is a central enzyme in nitrate assimilation and is localized in plastids. The present study concerns the regulation of the appearance of NIR in cotyledons of the mustard (Sinapis alba L.) seedling. It was shown that light exerts its positive control over the nitrate-mediated induction of NIR via the farred-absorbing form of phytochrome. Without nitrate the light effect cannot express itself; even though the light signal is accumulated in the cotyledons it remains totally cryptic in the absence of nitrate. Moreover, it was recognised that intact plastids are important in the control of the appearance of NIR. If the plastids are damaged by photooxidation the action of nitrate and phytochrome on NIR appearance is abolished. The appearance of nitrate reductase (NR; EC 1.6.6.1) responds similarly to photooxidative damage even though this enzyme is cytosolic. While the data strongly indicate that some plastidic signal is a prerequisite for the nitrate-induced and phytochrome-modulated appearance of NIR and NR, the possibility could not be ruled out that photooxidative damage affects the accumulation of NIR in the organelle.Abbreviations c continuous - D darkness - FR far-red light - NADP-GPD NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.1.13) - NF Norflurazon - NIR nitrite reductase (EC 1.7.7.1.) - NR nitrate reductase (EC 1.6.6.1) - Pfr phytochrome (far-red light obtained with RG9 glass filter - R red light - RG9-light long wavelenght far-red light obtained with RG9 glass filter - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - WL white light - WL_s strong white light (28 W m^-2)     

Signal storage in phytochrome action on nitrate-mediated induction of nitrate and nitrite reductases in mustard seedling cotyledons

Application of nitrate leads to an induction of nitrate reductase (NR; EC 1.6.6.1) and nitrite reductase (NIR; EC 1.7.7.1) in the cotyledons of dark-grown mustard (Sinapis alba L.) seedlings, and this induction can strongly be promoted by a far-red-light pretreatment — operating through phytochrome — prior to nitrate application. This light treatment is almost ineffective — as far as enzyme appearance is concerned — if no nitrate is given. When nitrate is applied, the stored light signal potentiates the appearance of NR and NIR in darkness, even in the absence of active phytochrome, to the same extent as continuous far-red light. This action of previously stored light signal lasts for approx. 12 h.Storage of the light signal was measured for NR and NIR. The process shows enzyme-specific differences. Storage occurs in the absence as well as in the presence of nitrate, i.e. irrespective of whether or not enzyme synthesis takes place. The kinetics of signal transduction and signal storage indicate that the formation and action of the stored signal are a bypass to the process of direct signal transduction. Signal storage is possibly a means of enabling the plant to maintain the appropriate levels of NR and NIR during the dark period of the natural light/dark cycle.Abbreviations cD continuous darkness - cFR continuous far-red light - D darkness - FR far-red light - NIR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.6.6.1) - Pfr phytochrome (far-red absorbing) - Pr phytochrome (red absorbing) - R red light - RG9-light long wavelength far-red light obtained with RG9 glass filter - - Ptot total phytochrome (Pr+Pfr) Professor Wilhelm Nultsch mit guten Wünschen zum 60. Geburtstag     

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO ₃ ^- in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH ₄ ⁺ or urea as nitrogen sources. Moreover, the presence of NH ₄ ⁺ did not abolish the NO ₃ ^- -dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO ₃ ^- but not by NH ₄ ⁺ or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

Induction of nitrate reductase, nitrite reductase, and glutamine synthetase isoforms in sunflower cotyledons as affected by nitrate, light, and plastid integrity

Summary We investigated the inducibility of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), and glutamine synthetase (GS; EC 6.3.1.2) isoforms in cotyledons of 7-day-old seedlings of sunflower (Helianthus annuus L.) in relation to light, nitrogen source (NO ₃ ^– , NO ₂ ^– or NH ₄ ⁺ ), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO ₂ ^– or NH ₄ ⁺ the synthesis of NiR and chloroplastic GS (GS₂) over the constitutive levels present in N-free-grown seedlings. In vivo inhibition of NR activity by tungsten application to seedlings and measurements of tissue NO ₃ ^– concentration indicate that NO ₃ ^– -dependent enzyme induction is elicited by NO ₃ ^– per se and not by a product of its assimilatory reduction, e.g., NO ₂ ^– or NH ₄ ⁺ . In the presence of NO ₃ ^– , light remarkably enhanced the appearance of NR, NiR, and GS₂, while the activity of the cytosolic GS isoform (GS₁) was adversely affected. Cycloheximide suppressed much more efficiently than chloramphenicol the light- and NO ₃ ^– -dependent increase of GS₂ activity, indicating that sunflower chloroplastic GS is synthesized on cytoplasmic 80S ribosomes. When the plastids were damaged by photooxidation in cotyledons made carotenoid-free by application of norflurazon, the positive action of light and NO ₃ ^– on the appearance of NR, NiR, and GS₂ isoform was greatly abolished. Therefore, it is suggested that intact chloroplasts are required for the inductive effect of light and NO ₃ ^– and/or for the accumulation of newly formed enzymes in the organelle.Abbreviations CAP chloramphenicol - CHX cycloheximide - GS glutamine synthetase - GS₁ cytosolic GS - GS₂ plastidic (chloroplastic) GS - NF norflurazon - NiR nitrite reductase - NR nitrate reductase     

Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

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.Abbreviations Chl chlorophyll - MC microcystin-LR - PEP-Case phosphoenolpyruvate carboxylase - SPS sucrose-phosphate synthase We are indebted to Madeleine Provot and Nathalie Hayes for excellent technical assistance. This work was funded by EEC Biotechnology Contract No. BI02 CT93 0400, project of technical priority, Network D — Nitrogen Utilisation and Efficiency.     

Factors involved in the coordinate appearance of nitrite reductase and glutamine synthetase in the mustard (Sinapis alba L.) seedling

The extent to which the appearances of nitrite reductase (NIR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) are coordinated was studied in mustard (Sinapis alba L.) seedlings. It was established by immunotitration that the increased activities of NIR and GS in the presence of light and nitrate can be attributed to the de-novo synthesis of enzyme protein. The bulk of the NIR and GS was found in the developing cotyledons. In the absence of nitrate in the growth medium there was no coordinate appearance of NIR and GS. While light strongly stimulated the appearance of GS, the level of NIR was hardly affected and remained low. On the other hand, in the presence of nitrate in the medium the appearances of NIR and GS were strictly coordinated, the GS level being considerably above that of NIR. It is argued that phytochrome-controlled synthesis of GS in the absence of nitrate is part of the mechanism to reassimilate ammonium liberated during proteolysis of storage protein and metabolism of the resulting amino acids, whereas the strictly coordinated synthesis in the presence of light and nitrate indicates the dominance of nitrate assimilation under these circumstances. The fact that the level of GS was always considerably above that of NIR appears to be a safety measure to prevent ammonium accumulation.Abbreviations FR standardized far-red light (3.5 W·m^–2), to drive the high-irradiance reaction of phytochrome - GS glutamine synthetase, EC 6.3.1.2 - NIR nitrite reductase, EC 1.7.7.1 This work was supported by Heidelberger Akademie der Wissenschaften (Forschungsstelle Nitratassimilation).     

Appearance of nitrite-reductase mRNA in mustard seedling cotyledons is regulated by phytochrome

Nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NIR, EC 1.7.7.1) are the key enzymes of nitrate reduction. It is well established that the appearance of these enzymes is “induced” by nitrate, and it is generally believed that NR is cytosolic while NIR is plastidic. In mustard (Sinapis alba L.) cotyledons we observed two isoforms of NIR (NIR₁ and NIR₂) using a chromato-focusing technique. Only one of them (NIR₂) disappeared when the plastids were damaged by photooxidation in the presence of Norflurazon. It is concluded that NIR₂ is plastidic while NIR₁ is extraplastidic and not affected by photooxidation of the plastids. Both isoforms appear to have the same molecular weight (60 kilodaltons, kDa). Two distinct translation products which could be immunoprecipitated with NIR antiserum produced against total NIR from mustard were observed which differed slightly in molecular weight (60 versus 63 kDa). The 63-kDa polypeptide was considered to be the precursor of NIR₂. While synthesis of NIR protein depended largely on nitrate, the levels of in-vitro-translatable NIR mRNAs were found to be either independent of nitrate and light (NIR₁) or controlled by phytochrome only (NIR₂). It appears that phytochrome strongly stimulates the level of mRNA while significant enzyme synthesis (NIR₂) takes place only in the presence of relatively large amounts of nitrate. Since an increased enzyme level was strictly correlated with an increase of immunoresponsive NIR protein it is improbable that activation of a precursor plays a role. Rather, it is concluded that, in situ, nitrate controls translation.     

Iron assimilation in plants: reduction of a ferriphytosiderophore by NADH:nitrate reductase from squash

NADH:nitrate reductase (EC 1.6.6.1) from squash (Cucurbita maxima Duch., cv. Buttercup) can catalyze the reduction of a ferriphytosiderophore from barley (Hordeum vulgare L. cv. Europa). Maximal activity occurs at pH 6, with an apparentK _m andV _max of 76 M and 21 nmol·min^-1·(mg protein)^-1, respectively. The ferriphytosiderophore strongly inhibits nitrate reduction catalyzed by nitrate reductase at the optimal pH for nitrate reduction, i.e. 7.5. On the contrary, nitrate is a poor inhibitor of ferriphytosiderophore reduction catalyzed by nitrate reductase at the optimal pH for this reaction, pH 6.0. Thus, squash has the potential to assimilate the iron from a ferriphytosiderophore synthesized by another plant.     

Regulation of nitrate reductase and phosphoenolpyruvate carboxylase activities in barley leaf protoplasts

Barley leaf protoplasts were incubated in light or darkness in the presence of various inhibitors, metabolites or weak acids/bases. Nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPCase) were rapidly extracted from the protoplasts and assayed under sub-optimal conditions, i.e. in the presence of Mg²⁺ and malate, respectively. Under these conditions changes in activities are thought to reflect changes in the phosphorylation states of the enzymes. The NR was activated by illumination to 90% of its maximal activity within 10 min. Photosynthetic electron transport appeared necessary for light activation of NR since activation was inhibited by the photosynthetic electron-transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and, additionally, an electron acceptor (HCO ₃ ^- ) was required. The PEPCase was also activated by light. However, this activation was not prevented by DCMU or lack of HCO ₃ ^- . Loading of protoplasts in the dark with a weak acid resulted in activation of both NR and PEPCase. For NR, full activation was completed within 5 min, whereas for PEPCase a slower, modest activation continued for at least 40 min. Incubation of protoplasts with a weak base also gave activation of PEPCase, but not of NR. On the contrary, base loading counteracted light activation of NR. Since several treatments tested resulted in the modulation of either NR or PEPCase activity, but not both, signal transduction cascades leading to changes in activities appear to be very different for the two enzymes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - DMO 5,5-dimethyl-2,4 oxazolidinedione - NR nitrate reductase - PEPCase Phosphoenolpyruvate carboxylase This work was supported by the Norwegian Research Council by a Grant to C.L: L.H.S. was supported by the Biotechnology and Biological Sciences Research Council.     

Utilization of nitrite and nitrate by dwarf bean

The uptake and conversion of NO ₂ ^- and the effect of NO ₂ ^- on the uptake and reduction of NO ₃ ^- were examined in N-depleted Phaseolus vulgaris L. Nitrite uptake at 0.1 mmol dm^-3 was against an electrochemical gradient and became constant after one or two initial phases. Steadystate uptake declined with increasing ambient NO ₂ ^- concentration (0–0.7 mmol dm^-3). In this concentration range root oxygen consumption was unaffected by NO ₂ ^- , indicating that the decrease of NO ₂ ^- uptake was not related to respiration. After 6 h NO ₂ ^- supply, about one-third of the absorbed NO ₂ ^- had accumulated, mainly in the root system. Oxidation of NO ₂ ^- to NO ₃ ^- was not observed. The apparent induction period for NO ₃ ^- uptake was about 6 h in control plants and 3.5 h in plants that were pretreated for 18 h with NO ₂ ^- . In contrast, the time course of NO ₂ ^- uptake was unaffected by pretreatment with NO ₃ ^- . Steadystate NO ₃ ^- uptake was less affected by NO ₂ ^- than was steady-state NO ₂ ^- uptake by NO ₃ ^- . Nitrate reductase activity (NRA) in leaves and roots was induced by both NO ₃ ^- and NO ₂ ^- . In roots, induction with NO ₂ ^- was faster than with NO ₃ ^- , but there was no difference in NRA after 5 h. Nitrite inhibited NRA in the roots of NO ₃ ^- -induced plants and thus seems to stimulate the induction, but not the activity of induced nitrate reductase. In view of the observed differences in time course and mutual competition, a common uptake mechanism for NO ₂ ^- and NO ₃ ^- seems unlikely. Expression of the NO ₂ ^- effect on the induction of NO ₃ ^- uptake required more time than the induction itself. We therefore conclude that NO ₂ ^- is not the physiological inducer of NO ₃ ^- uptake.Abbreviations NR(A) nitrate reductase (activity) - BM basal medium     

Ammonium and nitrate uptake in gap,generalist and understory species of the genus Piper

Summary We studied root net uptake of ammonium (NH ₄ ⁺ ) and nitrate (NO ₃ ^– ) in species of the genus Piper (Piperaceae) under high, intermediate and low photosynthetically active photon flux densities (PFD). Plants were grown hydroponically, and then transferred to temperature controlled (25° C) root cuvettes for nutrient uptake determinations. Uptake solutions provided NH ₄ ⁺ and NO ₃ ^– simultaneously (both) or separately (single). In the first experiment, seven species of Piper, from a broad range of rainforest light habitats ranging from gap to understory, were screened for mineral nitrogen preference (100 M NH ₄ ⁺ and/or 100 M NO ₃ ^– ) at intermediate PFD (100 mol m^–2 s^–1). Preference for NH ₄ ⁺ relative to NO ₃ ^– , defined as the ratio of NH ₄ ⁺ (both):NO ₃ ^– (both) net uptake, was higher in understory species than in gap species. Ammonium repression of NO ₃ ^– uptake, defined as the ratio of NO ₃ ^– (single): NO ₃ ^– (both) net uptake, was also higher in understory species as compared to gap species. In a second set of experiments, we examined the effect of nitrogen concentration (equimolar, 10 to 1000 M) on NH ₄ ⁺ preference and NH ₄ ⁺ repression of NO ₃ ^– net uptake at high (500 mol m^–2 s^–1) and low (50 mol m^–2 s^–1) PFD in a gap (P. auritum), generalist (P. hispidum) and understory species (P. aequale). All species exhibited negligible NH ₄ ⁺ repression of NO ₃ ^– net uptake at high PFD. At low PFD, NH ₄ ⁺ preference and repression of NO ₃ ^– net uptake occurred in all species (understory > generalist > gap), but only at intermediate nitrogen concentrations, i.e. between 10 and 200 M. Ammonium repression of net NO ₃ ^– uptake decreased or increased rapidly (in < 48 h) after transitions from low to high or from high to low PFD respectively. No significant diurnal patterns in NO ₃ ^– or NH ₄ ⁺ net uptake were observed.CIWDPB publication # 1130     

Pyridine nucleotide specificity and other properties of purified nitrate reductase from Chlorella variegata

Nitrate reductase (NR) (EC 1.6.6.2) from Chlorella variegata 211/10d has been purified by blue sepharose affinity chromatography. The enzyme can utilise NADH or NADPH for nitrate reduction with apparent K _m values of 11.5 M and 14.5 M, respectively. Apparent K _m values for nitrate are 0.13 mM (NADH-NR) and 0.14 mM (NADPH-NR). The diaphorase activity of the enzyme is inhibited strongly by parachloromercuribenzoic acid; NADH or NADPH protects the enzyme against this inhibition. NR proper activity of the enzyme is partially inactive after extraction and may be activated after the addition of ferricyanide. The addition of NAD(P)H and cyanide causes a reversible inactivation of the NR proper activity although preincubation with either NADH or NADH and ADP has no significant effect.Abbreviations NR Nitrate reductase - FAD Flavin-adenine dinucleotide - FMN Riboflavin 5-phosphate - p-CMB para-Chloromercuribenzoic - BV Benzyl viologen     

Relationships between external nitrate availability,nitrate uptake and expression of nitrate reductase in roots of barley grown in N-limited split-root cultures

Despite the large number of studies of nitrate metabolism in plants, it remains undetermined to what extent this key plant system is controlled by overall plant N nutrition on the one hand, and by the nitrate ion itself on the other hand. To investigate these questions, V _max for nitrate uptake (high-affinity range), and nitrate reductase (NR) mRNA and activity, were measured in roots of N-limited barley (Hordeum vulgare L. cv. Golf) grown under conditions of constant relative addition of nitrate, with the seminal roots split between two culture compartments. The total amount of nitrate added per unit time (0.09·d^-1) was distributed between the two root parts (subroots) in ratios of 1000, 982, 955, 9010, 8020, and 5050. These nitrate-addition ratios resulted in nitrate fluxes ranging from 0 to 23 mol nitrate·g^-1 DW root·h^-1, while the external nitrate concentrations varied between 0 and 1.2 M. The apparent V _max for net nitrate uptake showed saturation-type responses to nitrate flux maintained during preceding growth. The flux resulting in half-maximal induction of nitrate uptake was approximately 4 mol nitrate·g^-1 DW root·h^-1, corresponding to an external nitrate concentration of 0.7 M. The activity of NR and levels of NR mRNA did not saturate within the range of nitrate fluxes studied. None of the parameters studied saturated with respect to the steady-state external nitrate concentration. At the zero nitrate addition — the 0%-root — initial uptake activity as determined in short-term ¹⁵N-labelling experiments was insignificant, and NR activity and NR mRNA were not detectable. However, nitrate uptake was rapidly induced, showing that the 0%-root had retained the capacity to respond to nitrate. These results suggest that local nitrate availability has a significant impact on the nitrate uptake and reducing systems of a split-root part when the total plant nitrate nutrition is held constant and limiting.Abbreviation NR nitrate reductase This work was supported by the Lars Hierta Memory Foundation, the Royal Swedish Academy of Sciences, and by the Swedish Natural Science Research Council via project grants (to C.-M.L. and B.I.) and visiting scientist grant (to W.H.C.). We thank Mrs. Ellen Campbell for technical advice, and Mrs. Judith V. Purves, Long Ashton Research Station, Long Ashton, UK, for analyses of ¹⁵N-labelling in tissue samples.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme [C. Lillo et al. (2003) Plant J 35:566–573]. When cut leaves or roots of this line (S₅₂₁) were placed in darkness in a buffer containing 50 mM KNO₃, nitrite was excreted from the tissue at rates of 0.08–0.2 mol (g FW)^–1 h^–1 for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1–3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S₅₂₁, although 20–40 mol (g FW)^–1 nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S₅₂₁ also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S₅₂₁ was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.Abbreviations NR Nitrate reductase - NO Nitric oxide - Ser Serine - WT Wild type     

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 mol photons·m^–2 ·s^–1). This relationship disappeared at higher irradiance (450 mol 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 mol photons·m^–2·s^–1 than in those grown at 170 mol 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.Abbreviations Chl chlorophyll - N nitrogen - NR NADH-nitrate reductase - WT wild type     

Metabolic activators of spinach leaf nitrate reductase: Effects on enzymatic activity and dephosphorylation by endogenous phosphatases

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L.) leaves was inactivated in the dark and reactivated by light in vivo. When extracted from dark leaves, NR activity was lower and more strongly inhibited by Mg²⁺ relative to the enzyme extracted from leaves harvested in the light. When dark extracts were desalted at pH 6.5 and preincubated at 25° C prior to assay, enzyme activity (assayed either in the presence or absence of Mg²⁺) remained essentially constant, i.e. there was no spontaneous reactivation in vitro. However, addition of certain metabolites resulted in a time- and concentration-dependent activation of NR in vitro. Effective activators included inorganic phosphate (Pi), 5-AMP, and certain of its derivatives such as FAD and pyridine nucleotides (both oxidized and reduced forms). All of the activators increased NR activity as assayed in the absence of Mg²⁺, whereas some activators (e.g. Pi, 5-AMP and FAD) also reduced Mg²⁺ inhibition. The reduction of Mg²⁺ inhibition was also time-dependent and was almost completely prevented by a combination of okadaic acid plus KF, suggesting the involvement of dephosphorylation catalyzed by endogenous phosphatase(s). In contrast, the activation of NR (assayed minus Mg²⁺) was relatively insensitive to phosphatase inhibitors, indicating a different mechanism was involved. Compounds that were not effective activators of NR included sulfate, ribose-5-phosphate, adenosine 5-monosulfate, coenzyme A, ADP and ATP. We postulate that NR can exist in at least two states that differ in enzymatic activity. The activators appear to interact with the NR molecule at a site distinct from the NADH active site, and induce a slow conformational change (hysteresis) that increases NR activity (assayed in the absence of Mg²⁺). Possibly as a result of the conformational change caused by certain activators, the regulatory phospho-seryl groups are more readily dephosphorylated by endogenous phosphatases, thereby reducing sensitivity to Mg²⁺ inhibition. Preliminary results suggest that light/dark transitions in vivo may alter the distribution of NR molecules between the low- and high-activity forms.Abbreviations AP₅A P¹, P⁵-di(adenosine-5)pentaphosphate - DTT dithiothreitol - Mops 3-(N-morpholino)propanesulfonic acid - NR NADH:nitrate reductase - NRA nitrate reductase activity Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. This work was also supported in part by grants from the U.S. Department of Energy (Grant DE-AIO5-91 ER 20031) and USDA-NRI (Grant 93-373-5-9231). The authors thank Dr. W.M. Kaiser (Lehrstuhl Botanik I der Universität, Würzburg, Germany) for discussions and Dr. C. Lillo (Rogaland University Center, Stavanger, Norway) for sharing results prior to publication.