Effects of sodium on mineral nutrition in rose plants

The effects of sodium (Na⁺) ion concentration on shoot elongation, uptake of ammonium (NH₄⁺) and nitrate (NO₃^?) and the activities of nitrate reductase (NR) and glutamine synthetase (GS) were studied in rose plants (Rosa hybrida cv. “Lambada”). The results showed that shoot elongation was negatively correlated with sodium concentration, although no external symptoms of toxicity were observed. Nitrate uptake decreased at high sodium levels, specifically at 30 meq litre4 of sodium. As flower development was normal under high saline conditions, this could suggest that nitrogen was being mobilised from shoot and leaf reserves. Ammonium uptake was not affected by any of the salt treatments applied probably because it diffuses through the cell membrane at low concentrations. Nitrate reductase activity was reduced by 50% at 30 meq litre 1 compared with control treatment, probably due to a decrease in the free nitrate related to nitrate uptake pattern. None of the salt treatments used affected total leaf GS activity (both chloroplastic and cytosolic isoforms) or leaf NPK mineral contents. Nitrate reductase activity in leaves increased at 10 meq litre^?1 of sodium and GS activity in roots (cytosolic isoform only) followed the same pattern as NR. It is suggested that the activation of both enzymes at low salt level could be attributed to the beneficial effect of increased sulphur in the nutrient solutions.     

Response of Eggplant to Nitrogen Supply: Molybdenum-Nitrate Relationships

Greenhouse experiments were conducted in two years (1993–1994) with eggplants supplied with 1, 2 or 4 mM NH₄NO₃ as the N source in order to determine its influence on molybdenum (Mo) and nitrate (NO₃ ^–) content in leaf blades, petioles, and fruits as well as leaf nitrate reductase (NR) activity. The results reveal that 2 and 4 mM NH₄NO₃ altered shoot Mo distribution and thus affecting the NR activity.     

Nitrate Reductase Activity in an Eutrophic Reservoir duringthe Stratification Cycle

Nitrate reductase activity was measured during the stratification cycle of the eutrophic reservoir La Concepción. Nitrate was consumed in the epilimnion mainly through assimilatory reduction, and in the hypolimnion through dissimilatory reduction triggered by anoxic conditions. Nitrate reductase activity (NR) scaled to particulate nitrogen (PN) was positively correlated with the external nitrate concentration and the NO₃^–/NH₄⁺ ratio. Using NR as a tool for the assessment of the N cycle dynamics, it can be suggested that at the beginning of the stratification cycle, the phytoplanktonic community was mainly using NO₃^– as N source; and since N was not likely the limiting nutrient at that time of the year, NR was close to its maximum possible.     

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.Abbreviations Cyt c Rase cytochrome c reductase - ELISA enzyme-linked immunosorbent assay - FAD(H₂) flavin adenine dinucleotide (reduced form) - FMN(H₂) flavin mononucleotide (reduced form) - McAb monoclonal antibody - MV methyl viologen reduced form - NR nitrate reductase     

Nitrate regulation of nitrate uptake and nitrate reductase expression in barley grown at different nitrate:ammonium ratios at constant relative nitrogen addition rate

Barley (Hordeum vulgare L. cv. Golf) was cultured using the relative addition rate technique, where nitrogen is added in a fixed relation to the nitrogen already bound in biomass. The relative rate of total nitrogen addition was 0.09 day^?1 (growth limiting by 35%), while the nitrate addition was varied by means of different nitrate: ammonium ratios. In 3- to 4-week-old plants, these ratios of nitrate to ammonium supported nitrate fluxes ranging from 0 to 22 μmol g^?1 root dry weight h^?1, whereas the total N flux was 21.8 ± 0.25 μmol g^?1 root dry weight h^?1 for all treatments. The external nitrate concentrations varied between 0.18 and 1.5 μM. The relative growth rate, root to total biomass dry weight ratios, as well as Kjeldahl nitrogen in roots and shoots were unaffected by the nitrate:ammonium ratio. Tissue nitrate concentration in roots were comparable in all treatments. Shoot nitrate concentration increased with increasing nitrate supply, indicating increased translocation of nitrate to the shoot. The apparent V_max for net nitrate uptake increased with increased nitrate fluxes. Uptake activity was recorded also after growth at zero nitrate addition. This activity may have been induced by the small, but detectable, nitrate concentration in the medium under these conditions. In contrast, nitrate reductase (NR) activity in roots was unaffected by different nitrate fluxes, whereas NR activity in the shoot increased with increased nitrate supply. NR-mRNA was detected in roots from all cultures and showed no significant response to the nitrate flux, corroborating the data for NR activity. The data show that an extremely low amount of nitrate is required to elicit expression of NR and uptake activity. However, the uptake system and root NR respond differentially to increased nitrate flux at constant total N nutrition. It appears that root NR expression under these conditions is additionally controlled by factors related to the total N flux or the internal N status of the root and/or plant. The method used in this study may facilitate separation of nitrate-specific responses from the nutritional effect of nitrate.     

Nitrate reductase inactivator from Spirodela polyrhiza (L.) Schleiden

NADH:nitrate reductase (EC 1.6.6.1) activity in the crude extract from Spirodela polyrhiza was relatively labile in vitro. Inclusion of polyvinylpolypyrrolidone into the extraction medium had only a slight effect on the stability of the enzyme, whereas addition of 3 % casein, azocasein, or other proteins to the extraction medium greatly increased the nitrate reductase (NR) activity. Various protease inhibitors were tested for their ability to prevent the loss of NR activity in vitro. Iodoacetate and para-chloromercuric benzoate, the thiol-protease inhibitors, as well as pepstatin, the aspartic-protease inhibitor had no effect on stability of the nitrate reductase. EDTA had a slight stimulatory effect, whereas 5 mM o-phenantroline, another inhibitor of the metallo-proteases increased the activity of nitrate reductase. The highest enzyme activity was found in the presence of phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate both being serine-protease inhibitors. The protease-like inactivator was separated from Spirodela polyrhiza by ammonium sulfate fractionation and acid treatment (pH 4.0). After centrifugation the protein of inactivator in supernatant adjusted to pH 7.5 was removed. When this fraction was examined by electrophoresis in polyacrylamide which copolymerized with edestin, the protein of the nitrate reductase inactivator remained at the cathode. Fractions containing a protein of inactivator degraded casein to products soluble in trichloroacetic acid. Inhibition of the inactivator proteolytic activity by phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate but not by other reagents (thiol- and metallo-protease inhibitors) suggested the involvement of a serine residue at its active site. The inactivator fraction from Spirodela polyrhiza resulted in a loss of the nitrate reductase activity in crude extracts from both cucumber and corn seedlings. A biochemical nature a protein of the nitrate reductase inactivator from S. polyrhiza is discussed.     

Substrates regulate the phosphorylation status of nitrate reductase

The effect of substrates on the phosphorylation status of nitrate reductase (NR; EC 1.6.6.1) was studied. The enzyme was obtained from the first leaf of 7-day-old oat (Avena sativa L. cv. Suregrain) plants, grown in the light. When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg²⁺. NR sensitivity to Mg²⁺ remained unchanged when 10 mM nitrate was added to crude extracts after ATP. Addition of nitrate before or simultaneously with ATP slightly decreased Mg²⁺ inhibition of NR, which was strongly diminished in the presence of 10 mM NO₃^?+ 100 µM NADH. Incubation with NADH alone did not affect the enzyme's susceptibility to Mg²⁺ inhibition. When ammonium sulfate was added to crude extracts, NR was recovered in a 0-40% saturation fraction (F1). After incubation of F1 with ATP, the sensitivity of the enzyme to Mg²⁺ inhibition remained low, but it strongly increased after mixing F1 with a 45-60% saturation fraction (F2) suggesting that also in oats an additional factor (inactivating protein, IP), which probably binds to phospho-NR, would be required to keep the phosphorylated enzyme inactive in a +Mg²⁺ medium. Addition of 10 mM NO₃^?+ 100 µM NADH together with desalted F2 did not prevent Mg²⁺ inhibition suggesting that NO₃^? did not interfere with IP binding to phospho-NR. Again, incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg²⁺, After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 mM NO₃^? at 30°C. On the other hand, a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH. Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho-NR induced by the substrates. The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase- or the kinase-proteins. It has been reported that nitrate binding to the enzyme's active site induces conformational changes in the NR protein. We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.     

UV-B Radiation Mediated Alterations in the Nitrate Assimilation Pathway of Crop Plants 2. Kinetic Characteristics of Nitrite Reductase

Cowpea [Vigna unguiculata (L.) Walp. cv. Co 4] seedlings were subjected to a weighted irradiance of 3.2 W m^-2 s^-1 of biologically effective ultraviolet-B radiation (UV-B, 280–320 nm) and the changes in the kinetic and other characteristics of nitrite reductase (NiR) were recorded. The activity of NiR was hampered by 19 % under UV-B irradiation compared to the control. The UV-B treated plants required higher concentrations of nitrate for the induction of NiR synthesis than the controls. The NiR activity decay kinetics showed that the UV-B treatment significantly lowers the t_1/2 of the enzyme, thereby indicating a reduced rate of enzyme turnover. The comparison of kinetic characteristics of nitrate reductase (NR) and NiR under UV-B treatment showed that NiR was not so sensitive to UV-B radiation as NR. As shown by enzyme turnover rates, NiR extracted from plants irradiated by UV-B in situ was less sensitive to UV-B radiation than the enzyme extract subjected to in vitro UV-B irradiation. Though NiR was less damaged by UV-B treatment than NR, subtle changes occurred in its kinetic characteristics. This revised version was published online in June 2006 with corrections to the Cover Date.     

DIURNAL FLUCTUATION OF NITRATE REDUCTASE ACTIVITY IN THE MARINE RED ALGA GRACILARIA TENUISTIPITATA (RHODOPHYTA)1

The biochemical characteristics and diurnal changes in activity of the enzyme nitrate reductase (NR; EC 1.6.6.1) from the marine red alga Gracilaria tenuistipitata var. liui Zhang et Xia are described. Different assay conditions were tested to determine the stability of NR. The crude extract of G. tenuistipitata has a NR specific activity of 10.2 U.mg⁻¹, which is higher than the NR activities found for other algae, plants, and fungi. This NR is highly active at a slightly alkaline pH and is stable over a wide range of temperature, with an optimal activity at 20° C. The apical portions of the thallus contain 64.9 ± 6.6% of the total NR specific activity. The apparent Michaelis-Menten (K_m) constant found for KNO₃ was 197 μM, and it was 95 μM for NADH. The NR from G. tenuistipitata can be included in the NADH-specific group, because no activity was found when NADPH was used as an electron donor. In extracts of algae grown under either continuously dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at the middle of the light phase, when activity is 30-fold higher than during the night phase.     

Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants

Regulation of nitrate reductase (NR) by reversible phosphorylation at a conserved motif is well established in higher plants, and enables regulation of NR in response to rapid fluctuations in light intensity. This regulation is not conserved in algae NR, and we wished to test the evolutionary origin of the regulatory mechanism by physiological examination of ancient land plants. Especially a member of the lycophytes is of interest since their NR is candidate for regulation by reversible phosphorylation based on sequence analysis. We compared Selaginella kraussiana, a member of the lycophytes and earliest vascular plants, with the angiosperm Arabidopsis thaliana, and also tested the moss Physcomitrella patens. Interestingly, optimization of assay conditions revealed that S. kraussiana NR used NADH as an electron donor like A. thaliana, whereas P. patens NR activity depended on NADPH. Examination of light/darkness effects showed that S. kraussiana NR was rapidly regulated similar to A. thaliana NR when a differential (Mg²⁺ contra EDTA) assay was used to reveal activity state of NR. This implies that already existing NR enzyme was post-translationally activated by light in both species. Light had a positive effect also on de novo synthesis of NR in S. kraussiana, which could be shown after the plants had been exposed to a prolonged dark period (7 days). Daily variations in NR activity were mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation of NR in S. kraussiana was inhibited by 3-(3,4-dichlorophenyl)-1*1-dimethylurea (DCMU), an inhibitor of photosynthesis and stomata opening. Evolutionary, a post-translational control mechanism for NR have occurred before or in parallel with development of vascular tissue in land plants, and appears to be part of a complex mechanisms for coordination of CO₂ and nitrogen metabolism in these plants.     

Ammonium can stimulate nitrate and nitrite reductase in the absence of nitrate in Clematis vitalba

Nitrogen assimilation was studied in the deciduous, perennial climber Clematis vitalba. When solely supplied with NO₃^– in a hydroponic system, growth and N-assimilation characteristics were similar to those reported for a range of other species. When solely supplied with NH₄⁺, however, nitrate reductase (NR) activity dramatically increased in shoot tissue, and particularly leaf tissue, to up to three times the maximum level achieved in NO₃^– supplied plants. NO₃^– was not detected in plant material that had been solely supplied with NH₄⁺, there was no NO₃^– contamination of the hydroponic system, and the NH₄⁺-induced activity did not occur in tobacco or barley grown under similar conditions. Western Blot analysis revealed that the induction of NR activity, either by NO₃^– or NH₄⁺, was matched by NR and nitrite reductase protein synthesis, but this was not the case for the ammonium assimilation enzyme glutamine synthetase. Exposure of leaf disks to N revealed that NO₃^– assimilation was induced in leaves directly by NO₃^– and NH₄⁺ but not glutamine. Our results suggest that the NH₄⁺-induced potential for NO₃^– assimilation occurs when externally sourced NH₄⁺ is assimilated in the absence of any NO₃^– assimilation. These data show that the potential for nitrate assimilation in C. vitalba is induced by a nitrogenous compound in the absence of its substrate and suggest that NO₃^– assimilation in C. vitalba may have a significant role beyond the supply of reduced N for growth.     

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA_max) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA_max and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA_max and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA_max and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA_max was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA_max in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR_max by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA_max. Received: 24 March 1999 / Accepted: 31 May 1999     

Organic nitrogen content and nitrate and nitrite reductase activities in tritordeum and wheat grown under nitrate or ammonium

Tritordeum is a fertile amphiploid derived from durum wheat (Triticum turgidum L. conv. durum) × a wild barley (Hordeum chilense Roem. et Schultz.). The organic nitrogen content of tritordeum grain (34 mg g^-1 DW) was significantly higher than that of its wheat parent (25 mg g^-1 DW). Leaf and root nitrogen content became higher in tritordeum than in wheat after four weeks of growth, independently of the nitrogen source (either NO₃ ^- or NH₄ ⁺). Under NO₃ ^- nutrition, tritordeum generally exhibited higher levels of nitrate reductase (NR) activity than wheat. Nitrite reductase (NiR) levels were however lower in tritordeum than in its wheat parent. In NH₄ ⁺-grown plants, both NR and NiR activities progressively decreased in the two species, becoming imperceptible after 3 to 5 weeks of growth. Results indicate that, in addition to a higher rate of NO₃ ^- reduction, other physiological factors must be responsible for the greater accumulation of organic nitrogen in tritordeum grain.     

Role of light and CO2 fixation in the control of nitrate-reductase activity in barley leaves

Nitrate reductase (NR, NADH:nitrate oxidoreductase, EC 1.6.6.1) from barley (Hordeum vulgare L. cv. Hassan) leaves was inactivated during a light-dark transition, losing approx. 50% of activity after 30 min of darkness. The dark inactivation was reversed by illumination of the seedlings, the kinetics of reactivation being similar to those of inactivation. High extractable NR activity and significant differences between illuminated and darkened leaves were observed in media containing EDTA and inorganic phosphate (P_i). Addition of Ca²⁺ ions during extraction and assay decreased NR activity from illuminated and darkened leaves, enhancing the light-dark difference. While no clear correlation could be found between irradiance and NR activity, a hyperbolic correlation appeared between extractable NR activity and in-vivo rates of CO₂ fixation, indicating that NR activation follows saturation kinetics with respect to CO₂ fixation. Furthermore, hexoses and hexose-phosphates fed to the leaves via the transpiration stream protected against the dark-inactivation of NR. The results indicate that carbon-assimilation products are regulatory factors of NR activity in barley leaves, mediating both the light-dark modulation of NR and its dependence upon CO₂ fixation.     

Bottle gourd rootstock-grafting affects nitrogen metabolism in NaCl-stressed watermelon leaves and enhances short-term salt tolerance

The plant growth, nitrogen absorption, and assimilation in watermelon (Citrullus lanatus [Thunb.] Mansf.) were investigated in self-grafted and grafted seedlings using the salt-tolerant bottle gourd rootstock Chaofeng Kangshengwang (Lagenaria siceraria Standl.) exposed to 100 mM NaCl for 3 d. The biomass and NO₃⁻ uptake rate were significantly increased by rootstock while these values were remarkably decreased by salt stress. However, compared with self-grafted plants, rootstock-grafted plants showed higher salt tolerance with higher biomass and NO₃⁻ uptake rate under salt stress. Salinity induced strong accumulation of nitrate, ammonium and protein contents and a significant decrease of nitrogen content and the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT) in leaves of self-grafted seedlings. In contrast, salt stress caused a remarkable decrease in nitrate content and the activities of GS and GOGAT, and a significant increase of ammonium, protein, and nitrogen contents and NR activity, in leaves of rootstock-grafted seedlings. Compared with that of self-grafted seedlings, the ammonium content in leaves of rootstock-grafted seedlings was much lower under salt stress. Glutamate dehydrogenase (GDH) activity was notably enhanced in leaves of rootstock-grafted seedlings, whereas it was significantly inhibited in leaves of self-grafted seedlings, under salinity stress. Three GDH isozymes were isolated by native gel electrophoresis and their expressions were greatly enhanced in leaves of rootstock-grafted seedlings than those of self-grafted seedlings under both normal and salt-stress conditions. These results indicated that the salt tolerance of rootstock-grafted seedlings might (be enhanced) owing to the higher nitrogen absorption and the higher activities of enzymes for nitrogen assimilation induced by the rootstock. Furthermore, the detoxification of ammonium by GDH when the GS/GOGAT pathway was inhibited under salt stress might play an important role in the release of salt stress in rootstock-grafted seedlings.     

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U₂). Analysis with marker enzymes indicated that the U₂ fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L₁ first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U₂ second upper phase     

Activation and stabilization of nitrate reductase by NADH in wheat and maize

Preincubation of nitrate reductase (NR) extracted from wheat shoot tips with NADH in vitro, activated and stabilized activity at both O° and 25°. However, preincubation with potassium ferricyanide inactivated the NR in vitro. NADH also stabilized the NR activity in extracts from maize shoot tips. It was observed that NR from both wheat and maize was active at low temperatures.     

Nitrate reduction and compartmentation in tomato leaves. I. Nitrate accumulation in leaf sections in aqueous and gaseous medium

Nitrate pools in tomato ( Lycopersicon esculentum Mill. cv. Azes) leaf sections were estimated. Nitrite accumulation in aqueous medium was found to be an inadequate estimate of nitrate pools in tomato leaves. The main reason for the cessation of nitrite accumulation was not depletion of nitrate in the metabolic pool but rather a rapid decay of nitrate reductase (NR) activity as measured by nitrite accumulation in vivo and in vitro. Nitrate diffuses out of the tissue into the medium at a rate higher than the accumulation of nitrite in the tissue. Nitrate leakage from the tissue accelerates the loss of NR activity. Nitrite accumulation in leaf sections kept in an anaerobic gaseous atmosphere ceased earlier than in aqueous medium, at a time when NR activity was still relatively high. Measuring nitrite accumulation in gaseous atmosphere is preferable since NR is more stable and movements of nitrate between pools more restricted.