Stability of nitrate reductase and source of its reductant in Sorghum seedlings

Pre-incubation of nitrate reductase from Sorghum seedlings with NADH increased enzyme activity by 25%. Ferricyanide had no effect. NADH protected the enzyme from inactivation during storage. Malonate inhibited in vivo nitrate reduction in Sorghum leaves by 95%. The inhibitory effect of malonate was reversed by fumarate. Sodium fluoride in the presence of phosphate also inhibited in vivo nitrate reduction by 60%. It is suggested that NADH generated via the citric acid cycle is utilized for nitrate reduction in Sorghum seedlings.     

Fate of nitrate during initial nitrate utilization by nitrogen-depleted dwarf bean

The fate of nitrate and nitrogen-15 was followed during the apparent induction phase (6h) for nitrate uptake by N-depleted dwarf bean (Phaseolus vulgaris L. ev. Witte Krombek). Experiments were done with intact plants and with detached root systems. Qualitatively and quantitatively, xylem exudation from detached roots was a bad estimate of the export of NO^?₃ or NO^?₃-¹⁵N from roots of intact plants. In vivo nitrate reductase activity (NRA) agreed well with in situ reduction, calculated as the difference between uptake and accumulation in whole plants, provided NRA was assayed with merely endogenous nitrate as substrate (‘actual’ NRA). The majority (75%) of the entering nitrate remained unmetabolized. Both nitrate reduction and nitrate accumulation occurred predominantly in the root system. Some (< 25%) of the root-reduced nitrate-N was translocated to the shoot. Nitrate uptake occurred against the concentration gradient between medium and root cells, and probably against the gradient of the electro-chemical potential of nitrate. Part of the energy expended for NO^?₃ absorption came from the tops, since decapitation and ringing at the stem base restricted nitrate uptake.     

Age-dependent conversion of nitrate reductase to cytochrome c reductase species in barley leaf extracts

The stability of nitrate reductase (NR) in extracts from 4-, 5- and 6-day-old primary leaves of barley was examined. The half-time of loss of NR activity was found to be 358, 107 and 70 min, respectively. Bovine serum albumin (BSA) and phenylmethylsulphonylfluoride (PMSF) stabilized NR in extracts from 5- and 6-day-old primary leaves, but BSA was much more effective. The increased instability of NR with age correlated with increased conversion of the MW 203 000 NR complex to smaller NADH cytochrome c reductase (CR) species of MW 163 000, 61 000 and 40 000. The MW 163 000 CR species also possessed NR activity. BSA prevented and PMSF retarded the conversion of NR to the smaller CR species. The increased instability of NR in extracts from older tissue may be due to increased conversion of NR to smaller CR species. The ability of PMSF and BSA to stabilize NR and inhibit conversion of NR to the smaller CR species indicates that these phenomena are probably due to proteolytic degradation of NR. This suggestion is supported by the observation that trypsin cleaved NR to 3–4 S CR species and that cleavage was retarded by the presence of BSA. Endogenous proteinase attack at specific sites between domains of the barley NR complex may generate the CR species seen in barley extracts. The MW 40 000 CR species probably carries at least the FAD domain.     

Nitrate assimilation and nitrogen circulation in Austrian pine

Nitrate uptake, reduction and translocation were examined in 5-week-old Austrian pines ( Pinus nigra Arnold var. nigricans Host.) during exposure to 5 m M NaNO₃. The rate of nitrate uptake was linear during the 7 h light period. ¹⁵N-NO₃⁻ was detected in all parts of the pine except in the needles. By the 7th hour, 43% of the absorbed nitrate had been reduced, and this increased to 64% by the 24th hour. The major part of the total reduction occurred in the roots at this growth stage. Accumulation of ¹⁵N in reduced soluble and insoluble fractions was more prevalent in roots than in shoots. In the needles, the translocated nitrogen was mainly incorporated into the insoluble fraction. It is likely that most of the nitrogen from nitrate was transported from the roots to the aerial organs as organic nitrogen; however part of the upward nitrogen flux took place as nitrate ions.
An experiment in which an exposure for 24 h to 5 m M Na¹⁵NO₃ was followed by 13 days exposure to Na¹⁴NO₃ (pulse chase experiment) revealed a half time of about 1 day for depletion of root nitrate. A large part of this depletion was due to the loss of ¹⁵N-NO₃⁻ to the nutrient solution. The remaining pool of ¹⁵N-nitrate was partitioned between a metabolically inactive and an active pool. During the chase period, the simultaneous decrease of ¹⁵N-incorporation in the soluble N fraction and increase in the insoluble N fraction in different pine parts, particularly in the needles, suggested that protein synthesis occurred mostly in young tissues of the shoot and was the major sink of the newly absorbed ¹⁵N-NO₃.     

Greenhouse gas dynamics in boreal, littoral sediments under raised CO2 and nitrogen supply

SUMMARY 1. The effects of increasing CO₂ and nitrogen loading and of a change in water table and temperature on littoral CH₄, N₂O and CO₂ fluxes were studied in a glasshouse experiment with intact sediment cores including vegetation (mainly sedges), taken from a boreal eutrophic lake in Finland. Sediments with the water table held at a level of 0 or at ?15 cm were incubated in an atmosphere of 360 or 720 p.p.m. CO₂ for 18 weeks. The experiment included fertilisation with NO₃^– and NH₄⁺ (to a total 3 g N m^?2). 2. Changes in the water table and temperature strongly regulated sediment CH₄ and cCO₂ fluxes (community CO₂ release), but did not affect N₂O emissions. Increase in the water table increased CH₄ emissions but reduced cCO₂ release, while increase in temperature increased emissions of both CO₂ and CH₄. 3. The raised CO₂ increased carbon turnover in the sediments, such that cCO₂ release was increased by 16–26%. However, CH₄ fluxes were not significantly affected by raised CO₂, although CH₄ production potential (at 22 °C) of the sediments incubated at high CO₂ was increased. In the boreal region, littoral CH₄ production is more likely to be limited by temperature than by the availability of carbon. Raised CO₂ did not affect N₂O production by denitrification, indicating that this process was not carbon limited. 4. A low availability of NO₃^– did severely limit N₂O production. The NO₃^– addition caused up to a 100‐fold increase in the fluxes of N₂O. The NH₄⁺ addition did not increase N₂O fluxes, indicating low nitrification capacity in the sediments.     

FIELD ASSAYS FOR MEASURING NITRATE REDUCTASE ACTIVITY IN ENTEROMORPHA SP. (CHLOROPHYCEAE), ULVA SP. (CHLOROPHYCEAE), AND GELIDIUM SP. (RHODOPHYCEAE)1

In situ and in vitro nitrate reductase (NR) activity assays designed for use in the field on Enteromorpha sp., Ulva sp., and Gelidium sp. are described. In optimizing each assay, a variety of compounds and assay conditions were tested for their ability to extract NR and preserve its activity. Enteromorpha sp. had similar levels of in vitro NR activity after exposure to the in situ assay buffer, demonstrating that neither NR induction nor activation likely occurs during the in situ assay. Storing freshly collected Enteromorpha sp. led to a reduction in NR activity over time. However, the use of liquid nitrogen to freeze algal tissue on site and subsequent storage at ?80° C preserved NR activity and allowed for later laboratory use of the optimized in vitro assay. Application of the in situ and in vitro assays to stands of Enteromorpha sp., Ulva sp., and Gelidium sp. in the field consistently found NR activity. In situ NR activity over 9 consecutive days in January demonstrated that Enteromorpha sp. responds to increases in nitrate availability. The influence of light on diel patterns of in vitro NR activity in the field was demonstrated for the first time as well. For the three species tested, these two assays provide a reliable tool for field investigation of the interaction between environmental signals (e.g. nutrient levels) and physiological signals (e.g. tissue metabolite levels) on nitrate reduction.     

Simultaneous occurrence of denitrification and nitrate ammonification in sediments of the French Mediterranean Coast

Dissimilatory nitrate reductions in coastal marine sediment of Carteau Cove (French Mediterranean Coast) were studied between April 1993 and July 1994. Simultaneous determination of denitrification and dissimilatory nitrate reduction to ammonium was achieved by using a combination of acetylene blockage and 15N techniques. After short incubations (maximum 5 h), a part of 15N labelled nitrate added to the sediment was recovered as ammonium without incorporation in organic matter. The result indicate that a fraction of nitrate was reduced to ammonium by a dissimilatory mechanism instead of denitrifying. Denitrifying and nitrate ammonifying activities ranged from 0 to 19.8 μmol l-1 d-1 and from 2.3 to 83.2 μmol l-1 d-1, respectively. Denitrification rates were highest in early spring whereas nitrate ammonification were highest in fall. The recovery of nitrate reduced as N2O-N plus ammonium was between 40 and 100%, the highest nitrogen losses were recorded in July. Depending on the station and time of year denitrification accounted for between 0 and 43% of the total nitrate reduction whereas dissimilatory nitrate reduction to ammonium (DNRA) accounted for between 18 and 100%. The reduction rate data suggest that the pathway of nitrate reduction to ammonium may be important in coastal sediments. This revised version was published online in July 2006 with corrections to the Cover Date.     

The use of natural abundance of nitrogen isotopes in plant physiology and ecology

Nitrogen stable isotope (¹⁵N, ¹⁴N) natural abundance has been much less used than carbon isotopes (¹³C, ¹²C) in plant physiology and ecology. Analytical problems, the lower fractional abundance of ¹⁵N than of ¹³C in the biosphere, the greater complexity of the N cycle relative to the C cycle, and smaller expressed discriminations in nature, are contributing factors. The major N pools, globally, have different isotope signatures: atmospheric N₂ is ¹⁵N-depleted relative to organic N (including sedimentary N), a situation resulting from a greater expressed discrimination in the organic N to N₂ (via denitrification) reaction than of diazotrophy during accumulation of the reduced N. Essentially all of the enzymes except nitrogenase which transform N compounds show discrimination against ¹⁵N, although for glutamine synthetase, and the amination of 2-oxoglutarate and pyruvate, this is only seen in terms of NH₄⁺ rather than the true substrate, NH₃. Discrimination is expressed in various N interconversions within plants, leading to substantial differences in δ¹⁵N (up to 12‰) among N compounds and macroscopic plant parts. N isotope fractionation during assimilation of exogenous combined N is often much lower than that expected from studies of isolated enzymes due to processes which show very little discrimination, such as limitation by transport through aqueous solution and membranes. Application of ¹⁵N/¹⁴N discrimination studies to plant ecology have concentrated largely on distinguishing diazotrophy from N supplied from combined N, based on the lower ¹⁵N/¹⁴N in diazotrophs due to the higher ¹⁵N/¹⁴N of combined N sources not being offset by fractionation during uptake. While potentially very useful, a number of pitfalls are discussed in its ecological use in both terrestrial and aquatic systems. N isotope discrimination is also useful in tracking N through food webs, and hence, back to combined N sources for plants.