Elevated carbon dioxide increases nitrate uptake and nitrate reductase activity when tobacco is growing on nitrate, but increases ammonium uptake and inhibits nitrate reductase activity when tobacco is growing on ammonium nitrate

ABSTRACTTo assess how diurnal changes of nitrate reductase (NIA) expression in leaves interact with upstream and downstream processes during nitrate utilization, nitrate uptake, and nitrate and ammonium metabolism were investigated at several times during the diurnal cycle in wild‐type tobacco. Plants were grown hydroponically on 2 mM nitrate to exclude possible complications due to changes in the external availability of nitrate, and to allow nitrate uptake to be measured in the growth conditions. (a) In leaves, the NIA transcript decreases during the day and recovers at night, and NIA activity increases three‐fold during the first part and declines during the second part of the light period. Nitrate decreases during the day and recovers at night, ammonium, glutamine, glycine and serine increase during the day and decrease at night, and 2‐oxoglutarate increases three‐fold after illumination and decreases during the last part of the light period. The amplitudes of the diurnal changes are similar to or larger than in tobacco grown on high nitrate in sand. The transcript for plastid glutamine synthetase (GLN2) is low at the end of the night and increases during the day, and glutamine synthetase activity increases to a peak at the end of the day and decreases at night. (b) In the roots, transcript levels for the high affinity nitrate transporter (NRT2) increase in the day and decrease at night. Nitrate uptake is about 40% higher during the day than at night. (c) Comparison of the diurnal changes of the leaf metabolite pools with the rate of nitrate uptake allows diurnal changes in fluxes to be estimated. During the first part of the light, the rate of nitrate assimilation is about two‐fold higher than the rate of nitrate uptake, and also exceeds the rate at which reduced nitrogen is metabolized in the GOGAT pathway. The resulting decrease of leaf nitrate and accumulation of nitrogen in intermediates of ammonium metabolism and photorespiration represent about 40 and 15%, respectively, of the total nitrate that enters the plant in 24 h. Later in the diurnal cycle as NIA expression and activity decline, this imbalance is reversed. NRT2 expression and nitrate uptake remain relatively high, and nitrate taken up during the night is used to replenish the leaf nitrate pool. Increased GLN2 expression in leaves during the second part of the light period allows continued assimilation of ammonium released during photorespiration and remobilization of the reduced nitrogen that accumulated earlier in the diurnal cycle.

The efficiency of nitrate use by turfgrasses is likely related to its efficiency of absorption by roots and its rate of metabolism in roots and shoots. This study was conducted to quantify the relationship between nitrate uptake rate and nitrate reductase activity with N use efficiency in a cool‐season turfgrass. Six cultivars of Kentucky bluegrass (Poa pratensis L.), which differ markedly in field performance, were used to measure intraspecific variation in nitrate uptake, in vivo nitrate reductase activity of roots and leaves, and N use efficiency expressed as clipping mass per unit N in clippings. Companion field studies compared N use efficiency and metabolism among 14 Kentucky bluegrass cultivars established on an Enfield silt loam (Coarse loamy over sandy skeletal, mixed, mesic, Typic Dystrochrepts). Nitrate uptake rate was determined by an in situ nitrate depletion method. Nitrate reductase activity was assayed by an optimized in vivo method. Significant differences among cultivars were observed for nitrate absorption, nitrate reductase activity in roots and leaves, and N use efficiency. Ambient nitrate concentrations influenced these parameters and their intraspecific differences. Nitrate uptake and reduction were saturable at external nitrate concentrations in excess of 1 mM. Regression analyses demonstrated that nitrate reductase activity in roots and leaves was strongly influenced by nitrate uptake rate. Nitrogen use efficiency was negatively related to ambient nitrate levels, nitrate uptake rate and nitrate reductase activity, with nitrate reductase activity in leaves having the strongest negative effect on use efficiency. These results suggest that the efficiency of N use by Kentucky bluegrass may be increased by genetically altering nitrate reductase activity and its partitioning between roots and shoots.

The effect of preconditioning nitrogen source and growth rate on the interaction between nitrate and ammonium uptake was determined inThalassiosira pseudonana (Clone 3H). A new method, using cells on a filter (Parslow et al. 1985), allowed continuous measurement of uptake from 0.5 to 9 min after the addition of nitrate, ammonium, or both, with no variation in concentration during the course of the experiment. For each preconditioning N source and growth rate, a series of uptake experiments was conducted, including controls with only nitrate or only ammonium, and others with different combinations of concentrations of nitrate and ammonium. For the first time, preference for ammonium was separated from inhibition of nitrate uptake by ammonium. Ammonium was the preferred N source, i.e. if nitrate and ammonium were presented separately, ammonium uptake rates exceeded nitrate uptake rates. Preference for ammonium varied with both preconditioning N source and growth rate. Inhibition of nitrate uptake by ammonium, determined by comparing nitrate uptake in the presence and absence of ammonium, was observed at ammonium concentrations > 1µM, but was rarely complete. Inhibition of nitrate uptake by ammonium was less in the ammonium-limited culture than in the cultures growing on nitrate, but invariant with growth rate in the nitrate-grown cultures. Below 1µM ammonium, nitrate uptake was often stimulated and rates exceeded those in the controls without ammonium. Ammonium uptake was not inhibited by the presence of nitrate.T. pseudonana fits the classical view of the interaction between nitrate and ammonium uptake in some respects, such as preference for ammonium, and inhibition of nitrate uptake by ammonium concentrations > 1µM. However, at ammonium concentrations typical of most marine environments, nitrate uptake occurs at rapid rates. In other respects, N uptake inT. pseudonana deviates from the classical view in the following ways: (1) stimulation of nitrate uptake by low concentrations of ammonium; (2) lack of inhibition of nitrate uptake by ammonium at low nitrate concentrations; and (3) variation in preference and inhibition with preconditioning, which is markedly different for other species. Because of the apparent enormous species variation in the interaction between nitrate and ammonium uptake and the lack of detailed information for a variety of species, it is difficult to generalize about the effect of ammonium on nitrate uptake, especially in the field, where prior N availability and species composition are not usually addressed.

We examined the effect of pretreatments (18 h at 5 //mol dm-3) with abscisic acid, the ethylene-releasing substance 'Ethephon', gibberellic acid, indoleacetic acid, kinetin and zeatin on nitrate uptake and in vivo nitrate reductase activity (NRA) in roots of nitrogen-depleted Phaseolus vulgaris L. Nitrate uptake showed an apparent induction pattern with a steady state after about 6 h, in all treatments. The nitrate uptake rate after 6 h was unaffected or at most 30% lower after treatments with the plant growth regulators. Gibberellic acid, kinetin and zeatin induced substantial NRA in roots in the absence of nitrate, whereas Ethephon enhanced NRA only during nitrate nutrition. Kinetin-induced NRA (Ki-NRA) was maximal after a pretreatment at 1 //mol dm~3, and showed a lag phase of 6-8 h. Ki-NRA was additive to nitrate-induced NRA (NOj-NRA) for at least 24 h, independent of the induction sequence. After full induction, Ki-NRA approximated 20% of NO7-NRA. Abscisic acid counteracted the development of Ki-NRA, but not of NOj-NRA. Cycloheximide and tungstate were equally effective to suppress the development of nitrate reductase activity after supply of kinetin or NO3. Our data are consistent with the operation of two independent enzyme fractions (Ki-NRA and NO7-NRA) with apparently identical properties but with separate control mechanisms. The absence of major effects of plant growth regulators on the time-course and rate of nitrate uptake suggests that exogenous regulators, and possibly endogenous phytohormones are of minor importance for initial nitrate uptake. The differential effect of some regulators on nitrate uptake and root NRA furthermore indicates that the processes of uptake and reduction of NOj are not obligatory or exclusively coupled to each other.

Growth, Acetylene Reduction Activity, Nitrate Uptake and Nitrate Reductase Activity of Azolla caroliniana and Azolla pinnata at Varying Nitrate Levels

A basic tenet of nitrogen utilization in phytoplankton is that ammoniuln inhibits nitrate uptake. Consequently, it is generally believed that little or no nitrate uptake occurs at ammonium concentrations above ca 1 yM. A thorough review of field studies shows that the reduction of nitrate uptake rate in the presence of ammonium is rarely so severe, and that it is a highly variable phenomenon. To simplify quantification of the interaction between nitrate and ammonium uptake, it is proposed that it be divided into an indirect interaction, preference, and a direct effect, inhibition. In order to determine preference and inhibition it is necessary to measure uptake of each inorganic nitrogen source alone and in the presence of increasing concentrations of the other nitrogen source. Preference for ammonium uptake is manifested primarily in a higher V,,,,, and lower K, for ammonium uptake than for nitrate uptake and is accentuated by low light and low nitrogen availability. However, although ammonium is the preferred nitrogen source for uptake, growth rates on nitrate usually equal or exceed those on ammonium. Inhibition of nitrate uptake by ammonium is much more variable, but when separated from preference is less extreme. It is also enhanced by low light, but unlike preference, i t is greater when phytoplankton are N sufficient. Species differences are apparent for both preference and inhibition, but there are only enough data for preference to determine how it varies among algal groups. Finally, there are reports of low concentrations of ammonium stimulating nitrate uptake and of nitrate inhibiting ammonium uptake. Such unexpected interactions along with variations in preference and inhibition with species composition and environmental conditions may account for the variability observed in field studies and will not be explainable or predictable until more is known about the underlying biochemical mechanisms. Even though it is not possible at present to model nitrate uptake accurately because of uncertainty about the interaction between ammonluln and nitrate uptake, it is quite evident that the simplistic view that nitrate uptake is reduced to zero if ammonium exceeds 1 1iM would often result in large underestimates of nitrate uptake and new production.

The uptake of nitrate and ammonium by a terrestial (Bromheadia finlaysonia) and an epiphytic (Dendrobium hybrid) orchid in solution culture has been studied. The rates of nitrate and ammonium were relatively linear, with higher rate of uptake for ammonium. The rates of nitrate uptake in terrestial and epiphytic orchids were 0.4 and 0.9 μmole gm fw-1 hr-1 respectively and they were considerably lower than those of most major crops. SEM studies show that the velamen of Bromheadia was 2 cells thick whereas that of Dendrobium was 8-10 cells thick. It is unlikely that the velamen is the major factor in restricting influx of nitrate or ammonium. Nitrate reductase (NR) and glutamine synthetase (GS) were present in roots and leaves of both orchids. NR was high in roots but low in leaves. The reverse was for GS. The activities of NR and GS was low but high enough to account for the rate of nitrate or ammonium uptake. It appears that the movement of ions across the transfer junction at the exodermis plays a major regulatory role in ion uptake by orchid root.

Diurnal changes in carbohydrates and nitrate reductase (NR) activity were compared in tobacco (Nicotiana tabacum. L.cv. Gatersleben) plants growing in a long (18 h light/6 h dark) and a short (6 h light/18 h dark) day growth regime, or after short-term changes in the light regime. In long-day-grown plants, source leaves contained high levels of sugars throughout the light and dark periods. In short-day-grown plants, levels of sucrose and reducing sugars were very low at the end of the night and, although they rose during the light period, remained much lower than in long days and declined to very low levels again by the middle of the night. Starch accumulated more rapidly in short-day-than long-day-grown plants. Starch was completely remobilised during the night in short days, but not in long days. A single short day/long night cycle sufficed to stimulate starch accumulation during the following light period. In long-day-grown plants, the Nia transcript level was high at the end of the night, decreased during the day, and recovered gradually during the night. In short-day-grown plants, the Nia transcript level was relatively low at the end of the night, decreased to very low levels at the end of the light period, increased to a marked maximum in the middle of the night, and decreased during the last 5 h of the dark period. In long-day-grown plants, NR activity in source leaves rose by 2- to 3-fold in the first part of the light period and decreased in the second part of the light period. In short-day-grown plants, NR activity was low at the end of the night, and only increased slightly after illumination. Dark inactivation of source-leaf NR was partially reversed in long-day-grown plants, but not in short day-grown plants. In both growth regimes, mutants with one instead of four functional copies of the Nia gene had a 60% reduction in maximum NR activity in the source leaves, compared to wild-type plants. The diurnal changes in NR activity were almost completely suppressed in the mutants in long days, whereas the mutants showed similar or slightly larger diurnal changes than wild-type plants in short days. When short-day-grown plants were transferred to long-day conditions for 3 d, NR activity and the diurnal changes in NR activity resembled those in long-day-grown plants. Phloem export from source leaves of short-day-grown plants was partially inhibited by applying a cold-girdle for one light and dark cycle. The resulting increase in leaf sugar was accompanied by an marked increase in the Nia transcript level and a 2-fold increase in NR activity at the end of the dark period. When wild-type plants were subjected to a single short day/long night cycle of increasing severity, NR activity in source leaves at the end of the night decreased when the endogenous sugars declined below about 3 mumol hexose (g FW)-1. In sink leaves in short-day conditions, sugars were higher and the light-induced rise in NR activity was much larger than in source leaves on the same plants. The source leaves of wild-type plants in short-day conditions contained very high levels of nitrate, very low levels of glutamine, low levels of total amino acids, and lower protein and chlorophyll, compared to long-day-grown plants. Plants grown in short days had relatively high levels of glutamate and aspartate, and extremely low levels of most of the minor amino acids in their source leaves at the end of the night. Illumination led to a decrease in glutamate and an increase in the minor amino acids. A single short day/long night cycle led to an increase in glutamate, and a large decrease in the minor acids at the end of the dark period, and reillumination led to a decrease in glutamate and an increase in the minor amino acids. It is proposed that sugar-mediated control of Nia expression and NR activity overrides regulation by nitrogenous compounds when sugars are in short supply, resulting in a severe inhibition of nitrate assimilation. It is also proposed that su

Differential regulatory role of nitric oxide in mediating nitrate reductase activity in roots of tomato (Solanum lycocarpum).

Most Vaccinium species, including V. corymbosum, have strict soil requirements for optimal growth, requiring low pH, high iron, and nitrogen, primarily in the ammonium form. V. arboreum is a wild species adapted to high pH, low iron, nitrate-containing soils. This broader soil adaptation in V. arboreum may be related to increased efficiency of iron or nitrate uptake/assimilation compared with cultivated Vaccinium species. To test this, nitrate and iron uptake, and nitrate reductase (NR) and ferric chelate reductase (FCR) activities were compared in two Vaccinium species, V. arboreum and the cultivated V. corymbosum. Plants were grown hydroponically for 15 weeks in either 1.0 or 5.0 mm NO3 with 0.09 mm Fe. Root FCR activity was greater in V. arboreum compared with V. corymbosum, especially at the lower external nitrate concentration. However, this was not reflected in differences in iron uptake. Nitrate uptake and root NR activity were greater in V. arboreum compared with V. corymbosum. The lower nitrate uptake and assimilation in V. corymbosum was reflected in decreased plant dry weight compared with V. arboreum. V. arboreum appears to be more efficient in acquiring nitrate compared with V. corymbosum, possibly due to increased NR activity, and this may partially explain the wider soil adaptation of V. arboreum.

Anodic oxidation of coumaric acid led to the inhibition of the process at the electrode due to a film which was formed after one-electron oxidation of the acid to phenoxy radical.By contrast, caffeic acid is oxidized in two steps-the phenoxy radical is formed in the first step, quinone in the second step. The inhibition of nitrate uptake by coumaric and caffeic acids is dependent on their concentration. 10-4 M eaffeic acid totally inhibited nitrate uptake and the growth ofNicotiana tabacum cell suspension. 10-6 M caffeic acid markedly inhibited nitrate uptake especially in the first three days after inoculation. 10-6 M coumaric acid did not affect nitrate uptake and nitrate reductase activity, 10-4 M coumaric acid inhibited nitrate uptake by day two after inoculation. Nitrate reductase synthesis correlated with the inhibition of nitrate uptake. Differential effects of coumaric and caffeic acids are explained on the basis of different products of their electrochemical oxidation.

The possibility that the primary effect of the toxic insecticide trichlorfon is an inhibition of nitrate uptake in cyanobactena has been investigated. A drastic reduction in the rate of uptake is detected 3 h after the addition of the insecticide to batch cultures of nabaena PCC 7119. The dose-response curves indicate a relationship between the degree of inhibition of nitrate uptake and the reduction of chlorophyll content and growth. Nitrate reductase (ferredoxin : nitrate reductase, EC 1.7.99.4) activity is also lowered as a result of insecticide action. When Anabaena PCC 7119 cells are grown with ammonium as a source of combined nitrogen, trichlorfon reduces the rate of ammonium uptake. The rate of uptake of both nitrate and ammonium is restored upon washing the cells. Ultrastructural analysis of Anabaena nitrate-grown cells shows that trichlorfon does not damage thylakoid membranes, but brings about the accumulation of enlarged cyanophycin granules and the increase of carboxysome number. Nitrate uptake rate and chlorophyll and phycobiliprotein contents are also reduced by insecticide treatment in the cyanobacteria Synechococcus UAM 211, GloeothecePCC 6501, Plectonema calothricoides, Nostoc UAM 205 and Chlorogloeopsis PCC 6912. These results are consistent with the inhibition of nitrate uptake due to weak adsorption of trichlorfon to the plasmalemma being the main effect of the insecticide on cyanobacterial metabolism.

Nitrogen uptake by tropical orchids

Nitrate Uptake and Reduction by Durum Wheat (Triticum turgidum) and Tritordeum (Hordeum chilense x Triticum turgidum)

Nitrate uptake, nitrate reductase distribution and their relation to proton release in five nodulated grain legumes.