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     

Inhibition of N2 fixation by white clover (Trifolium repens L.) at low concentrations of NO inf3 sup− in flowing solution culture

The impact of sustained low external concentrations of NO ₃ ^– (0, 10, 100 and 1000 mmol m^–3) on plant growth and the relative acquisition of N through N₂ fixation and NO ₃ ^– uptake by established, nodulated white clover (Trifolium repens L. cv. Blanca) was studied over 28 days in flowing solution culture. Nitrogen fixation was measured by N difference and ¹⁵N dilution methods. Plants supplied with NO ₃ ^– achieved higher relative growth rates (% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGabmiEayaara% aaaa!3702!\[\bar x\]=0.091 d^–1) compared with control plants dependent on N₂ fixation (0.073 d^–1). Nitrate plants showed progressive increases in shoot: root d.w. ratios from 4 to 6.5–7.6 between days 0–28, compared with 5.1 on day 28 for control plants. Increases in both nodule d.w. and numbers per plant were inhibited after day seven at all concentrations of NO ₃ ^– . The severity of inhibition of N₂ fixation increased with increasing NO ₃ ^– concentration and with time. The total amounts of N₂ fixed per plant between days 0–7 after supplying 10, 100 and 1000 mmol m^–3 NO ₃ ^– , respectively, were 37–39, 28–30 and 0–13%, of the total N acquired. Between days 7–28 the proportional contributions of N₂ fixation to total N acquisition declined to 3, 0.5 and 0%, respectively, in these treatments. The corresponding mean specific rates of N₂ fixation between days 0–7 were, respectively, 5.4, 3.2, and 2.0 mmol N d^–1 g^–1 nodule d.w., compared with 7.9 mmol N d^–1 g^–1 nodule d.w. for zero NO ₃ ^– plants. There was no evidence of a transitory increase in N₂ fixation following the addition of NO ₃ ^– , even at the lowest supply concentration.     

Loss of quantum yield in extremely low light

It has generally been assumed that the photosynthetic quantum yield of all C₃ plants is essentially the same for all unstressed leaves at the same temperature and CO₂ and O₂ concentrations. However, some recent work by H.C. Timm et al. (2002, Trees 16:47–62) has shown that quantum yield can be reduced for some time after leaves have been exposed to darkness. To investigate under what light conditions quantum yield can be reduced, we carried out a number of experiments on leaves of a partial-shade (unlit greenhouse)-grown Coleus blumei Benth. hybrid. We found that after leaves had been exposed to complete darkness, quantum yield was reduced by about 60%. Only very low light levels were needed for quantum yield to be fully restored, with 5 mol quanta m^–2 s^–1 being sufficient for 85% of the quantum yield of fully induced leaves to be achieved. Leaves regained higher quantum yields upon exposure to higher light levels with an estimated time constant of 130 s. It was concluded that the loss of quantum yield would be quantitatively important only for leaves growing in very dense understoreys where maximum light levels might not exceed 5 mol quanta m^–2 s^–1 even in the middle of the day. Most leaves, even in understorey conditions, do, however, experience light levels in excess of 5 mol quanta m^–2 s^–1 over periods where they obtain most of their carbon so that the loss of quantum yield would affect total carbon gain of those leaves only marginally.Abbreviations FBPase Fructose-1,6-bisphosphatase - RuBP Ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase     

Quantification of N2-fixation and survival of inoculated diazotrophs associated with roots of Kallar grass

White clover plants were grown for 97 days under two temperature regimes (20/15°C and 8/5°C day/night temperatures) and were supplied with either small amounts (a total of 80 mg N pot^–1) of ammonium (NH ₄ ⁺ ) or nitrate (NO ₃ ^– ) nitrogen, or received no mineral N and relied on N₂ fixation. Greatest growth and total leaf area of clover plants occurred in N₂ fixing and NO ₃ ^– -fed plants grown at 20/15°C and poorest growth occurred in NH ₄ ⁺ -fed plants grown at 8/5°C. Nodule mass per plant was greater at 8/5°C due to increased nodule numbers rather than increased dry weight per nodule. This compensated to some extent for the reduced N₂-fixing activity per unit dry weight of nodule tissue found at the low growth temperature up to 116 d after sowing, but thereafter both activity per nodule dry weight and activity per plant were greater at the low temperature. Highest nitrate reductase activity (NRA) per g fresh weight and total activity per leaf, petiole or root occurred in NO ₃ ^– -fed plants at 8/5°C. Low growth temperature resulted in a greater partitioning of total plant NRA to the roots of NO ₃ ^– -fed plants. The results are considered in relation to the use of N fertiliser in the spring under field conditions.     

Effect of tree distance on N2O and CH4-fluxes from soils in temperate forest ecosystems

Complete annual cycles of N₂O and CH₄ flux in forest soils at a beech and at a spruce site at the Höglwald Forest were followed in 1997 by use of fully automatic measuring systems. In order to test if on a microsite scale differences in the magnitude of trace gas exchange between e.g. areas in direct vicinity of stems and areas in the interstem region at both sites exist, tree chambers and gradient chambers were installed in addition to the already existing interstem chambers at our sites. N₂O fluxes were in a range of –4.6–473.3 g N₂O-N m^–2 h^–1 at the beech site and in a range of –3.7–167.2 g N₂O-N m^–2 h^–1 at the spruce site, respectively. Highest N₂O emissions were observed during and at the end of a prolonged frost period, thereby further supporting previous findings that frost periods are of crucial importance for controlling annual N₂O losses from temperate forests. Fluxes of CH₄ were in a range of +10.4––194.0 g CH₄ m^–2 h^–1 at the beech site and in a range of –4.4––83.5 g CH₄ m^–2 h^–1 at the spruce site. In general, both N₂O-fluxes as well as CH₄-fluxes were higher at the beech site. On a microsite scale, N₂O and CH₄ fluxes at the beech site were highest within the stem area (annual mean: 49.6±3.3 g N₂O-N m^–2 h^–1; –77.2±3.1 g CH₄ m^–2 h^–1), and significantly lower within interstem areas (18.5±1.4 g N₂O-N m^–2 h^–1; –60.2±1.8 g CH₄ m^–2 h^–1). Significantly higher values of total N, C and pH in the organic layer, as well as increased soil moisture, especially in spring, in the stem areas, are likely to contribute to the higher N₂O fluxes within the stem area of the beech. Also for the spruce site, such differences in trace gas fluxes could be demonstrated to exist (mean annual N₂O emission within (a) stem areas: 9.7±0.9 g N₂O-N m^–2 h^–1 and (b) interstem areas: 6.2±0.6 g N₂O-N m^–2 h^–1; mean annual CH₄ uptake within (a) stem areas: –26.1±0.6 g CH₄ m^–2 h^–1 and (b) interstem areas: –38.4±0.8 g CH₄ m^–2 h^–1), though they were not as pronounced as at the beech site.     

Nitrate induction in spruce: an approach using compartmental analysis

Using ¹³NO ₃ ^– -efflux analysis, the induction of nitrate uptake by externally supplied nitrate was monitored in roots of intact Picea glauca (Moench) Voss. seedlings over a 5-d period. In agreement with our earlier studies, efflux analysis revealed three compartments, which have been identified as surface adsorption, apparent free space, and cytoplasm. While induction of nitrate uptake was pronounced, NO ₃ ^– fluxes in induced plants were decidedly lower and the induction response was slower than in other species. Influx rose from 0.1 mol·g^–1·h^–1 (measured at 100 M [NO ₃ ^– _o) in uninduced plants to a maximum of 0.5 mol·g^–1h^–1 after 3 d of exposure to 100 M [NO ₃ ^– _o and declined to 0.3–0.4 mol·g^–1h^–1 at the end of the 5-d period. Efflux remained relatively constant around 0.02-0.04 mol·g^–1h^–1, but its percentage with respect to influx declined from initially high values (around 30%) to steady-state values of 4–7%. Cytoplasmic [NO ₃ ^– ] ranged from the low micromolar in uninduced plants to a maximum of 2 mM in plants fully induced at 100 M [NO ₃ ^– ]_o. In-vivo root nitrate reductase activity (NRA) was measured over the same time period, and was found to follow a similar pattern of induction as influx. The maximum response in NRA slightly preceded that of influx. It increased from 25 nmol·g^–1·h^–1 without prior exposure to NO ₃ ^– to peak values around 150 nmol· g^–1h^–1 after 2 d of exposure to 100 M [NO ₃ ^– ]_o. Subsequently, NRA declined by about 50%. The dynamics of flux partitioning to reduction, to the vacuole, the xylem, and to efflux during the induction process are discussed.The research was supported by an Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. J. Vidmar for essential assistance in experiments.     

Growth and respiration ofBradyrhizobium japonicum USDA 143 with nitrous oxide as the terminal electron acceptor

Bradyrhizobium japonicum USDA 143 grew chemoorganotrophically under anoxic conditions with exogenous N₂O as the sole terminal electron acceptor. Cell growth and dissimilatory N₂O reduction were significantly inhibited by C₂H₂ when either N₂O or N₂O plus NO ₃ ^– served as terminal electron acceptor(s). Reduction of N₂O accounted for 20% of the energy for cell growth in cultures supplied with NO ₃ ^– as the terminal electron acceptor. Nitrous oxide was produced stoichiometrically in cultures containing NO ₃ ^– and C₂H₂, but cell growth was proportionately reduced when compared with cultures supplied with an equal amount of NO ₃ ^– . Exogenous N₂O delayed the reduction of NO ₃ ^– in cultures supplied with both electron acceptors. Direct amperometric monitoring of N₂O respiration showed a specific activity of 0.082±0.004 moles N₂O/min/mg cell protein, and azide inhibited cell respiration.     

Application of N-15 dilution for simultaneous estimation of nitrification and nitrate reduction in soil-water columns

Nitrification and denitrification rates were estimated simultaneously in soil-floodwater columns of a Crowley silt loam (Typic Albaqualfs) rice soil by an¹⁵N isotopic dilution technique. Labeled NO ₃ ^– was added to the floodwater of soil-water columns, half were treated with urea fertilizer. The (NO ₃ ^– +NO ₂ ^– )–N and (NO ₃ ^– +NO ₂ ^– )–N concentrations in the floodwater were measured over time and production and reduction rates for NO ₃ ^– calculated. Nitrate reduction in the urea amended columns averaged 515 mol N m^–2h^–1 and nitrification averaged 395 mol N m^–2h^–1 over the 35–153 d incubation. The nitrification rate for 4–19 d sampling period (1,560 mol N m^–2h^–1) in the urea amended columns was almost 9 times greater than the reduction rate (175 mol N m^–2h^–1) over the same period. Without the addition of urea the NO ₃ ^– production rate averaged 32 mol N m^–2h^–1 and reduction 101 mol N m^–2h^–1.     

Short-term studies of NO 3 − uptake in Pisum using 13NO 3 −

Influx, efflux and net uptake of NO ₃ ^– was studied in Pisum sativum L. cv. Marma in short-term experiments where ¹³NO ₃ ^– was used to trace influx. The influx rate in N-limited plants was similar both during net uptake at external concentrations of around 50 M, and at low external NO ₃ ^– concentrations (4–6 M) when net uptake was practically zero. Efflux could be inferred from discrepancies between influx and net uptake but was never very high in the N-limited plants during net uptake. Close to the threshold concentration for not NO ₃ ^– uptake, efflux was high and equalled influx. Thus, the threshold concentration can be regarded as a NO ₃ ^– compensation point. The inclusion of NH ₄ ⁺ in the outer medium decreased influx by about 40% but did not significantly affect efflux. The roles of NO ₃ ^– fluxes and nitrate-reductase activity in regulating/limiting NO ₃ ^– utilization are discussed.Abbreviations DW dry weight - FW fresh weight - R_N relative nitrogen addition rate     

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     

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO ₃ ^– or NO ₂ ^– upon initial exposure to these anions. Ability of the plants to take up NO ₃ ^– and NO ₂ ^– at high rates from the beginning was induced by a pretreatment with NO ₃ ^– . Nitrite also acted as inducer of the NO ₂ ^– -uptake system. The presence of cycloheximide during NO ₃ ^– -pretreatment prevented the subsequent uptake of NO ₃ ^– and NO ₂ ^– , indicating that both uptake systems are synthesized de novo when plants are exposed to NO ₃ ^– . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO ₃ ^– and NO ₂ ^– . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO ₃ ^– and NO ₂ ^– uptake as a function of external anion concentration exhibited saturation kinetics. The calculated K_m values for NO ₃ ^– and NO ₂ ^– uptake were 45 and 23 M, respectively. Rates of NO ₃ ^– uptake were four to six times higher than NO ₃ ^– -reduction rates in roots. In contrast, NO ₂ ^– -uptake rates, found to be very similar to NO ₃ ^– -uptake rates, were much lower (about 30 times) than NO ₂ ^– -reduction rates. Removal of oxygen from the external solution drastically suppressed NO ₃ ^– and NO ₂ ^– uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO ₃ ^– and NO ₂ ^– into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI cycloheximide - NEM N-ethylmaleimide - NiR nitrite reductase - NR nitrate reductase - pHME p-hydroxymercuribenzoate This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance.     

Simultaneous gas exchange and fluorescence measurements indicate differences in the response of sunflower,bean and maize to water stress

Gas exchange and fluorescence measurements of attached leaves of water stressed bean, sunflower and maize plants were carried out at two light intensities (250 mol quanta m^-2s^-1 and 850 mol quanta m^-2s^-1). Besides the restriction of transpiration and CO₂ uptake, the dissipation of excess light energy was clearly reflected in the light and dark reactions of photosynthesis under stress conditions. Bean and maize plants preferentially use non-photochemical quenching for light energy dissipation. In sunflower plants, excess light energy gave rise to photochemical quenching. Autoradiography of leaves after photosynthesis in ¹⁴CO₂ demonstrated the occurrence of leaf patchiness in sunflower and maize but not in bean. The contribution of CO₂ recycling within the leaves to energy dissipation was investigated by studies in 2.5% oxygen to suppress photorespiration. The participation of different energy dissipating mechanisms to quanta comsumption on agriculturally relevant species is discussed.Abbreviations F_o minimal fluorescence - F_m maximal fluorescence - F_p peak fluorescence - g leaf conductance - P_N net CO₂ uptake - q_N coefficient of non-photochemical quenching - q_P coefficient of photochemical quenching     

Diurnal changes in nitric oxide emissions from conventional tillage and pasture sites in the Amazon Basin: influence of soil temperature

We have investigated a subset of restoration practices applied to a degraded pasture at Fazenda Nova Vida, a 22000 ha cattle ranch in Rond^onia, Brazil. Nitric oxide (NO) and carbon dioxide (CO₂) emissions from soils were measured in conventional tillage and current pasture sites to assess N and C losses. Mean daily NO emissions from tilled plots were at least twice those from the pasture. Nitric oxide emissions from the tilled sites showed a strong diurnal pattern, while those from the pasture sites did not. Mean daytime NO emissions from the tilled sites were 9.7 g NO-N m^–2 h^–1, while mean nighttime emissions were 29.7 g NO-N m^–2 h^–1. In the pasture sites, NO emissions were 7.6 g NO-N m^–2 h^–1 during the day, and 7.7 g NO-N m^–2 h^–1 at night. Surface soil temperature was a good inverse predictor (r ²=0.75) of NO emissions from the tilled sites. Carbon dioxide emissions from the tilled sites were generally larger than CO₂ emissions from the pasture sites. The mean CO₂ emission rate from the tilled sites was 179 mg C m^–2 h^–1, while it was 123 mg C m^–2 h^–1 from the pasture sites. There was no distinct diurnal pattern for CO₂ emissions. We found that the very high temperatures measured at the soil surface in the tillage plots, in the range of 40–45°C, reduced the rate of NO emission. The reduction in NO emissions may be because of the sensitivity of autotrophic nitrifiers to high temperatures. This study provides insights on how land-use change may alter regional NO fluxes by exposing certain microbial communities to extreme environmental conditions. Future studies of NO emissions in tropical agricultural systems where soils are bare for extend periods need to make diurnal measurements or the daily fluxes will be substantially underestimated.     

Three years of continuous measurements of atmospheric ammonia concentrations over a forest stand at the Höglwald site in southern Bavaria

Over a period of 3 years (1995 – 1997), atmospheric ammonia (NH₃) concentrations were measured 3 m above a spruce stand using a continuous-flow annular denuder at the Höglwald site near Munich, Bavaria. The annual average ammonia concentration was between 2.2 and 2.9 g NH₃ m^–3. The highest hourly average values occurred at the end of each year. In December 1995 the peak value was achieved with 183 g NH₃ m^–3. More than 50% of the hourly average means of the ammonia concentration were lower than 2 g NH₃ m^–3 and only fewer than 5% of the hourly average concentrations higher than 10 g NH₃ m^–3. The ammonia concentration course indicated a pronounced diurnal variation, with higher concentrations in the late morning and lower concentrations during the night. Often a sudden increase of the ammonia concentration was detected in the early morning with first sun exposure of the spruce crown and sinking humidity, indicating a reemission of ammonia from the canopy to the atmosphere.     

Denitrification in a South Louisiana wetland forest receiving treated sewage effluent

Although denitrification has the potential to reduce nitrate (NO ₃ ^– ) pollution of surface waters, the quantification of denitrification rates is complex because it requires differentiation from other mechanisms and is highly variable in both space and time. This study first measured potential denitrification rates at a wetland forest site in south Louisiana before receipt of secondary wastewater effluent, and then, following 30 months of effluent application, landscape gradients of dissolved nitrate (NO ₃ ^– ) and nitrous oxide (N₂O) were measured. A computer model was developed to quantify N transformations. Floodwater NO ₃ ^– and N₂O concentrations were higher in the forest receiving effluent than in the adjacent control forest. Denitrification rates of NO ₃ ^– -amended soil cores ranged from 0.03–0.45 g N m^–2 d^–1 with an overall mean of 0.10 g N m^–2 d^–1. Effluent N is being applied at a rate of approximately 0.034 g N m^–2 d^–1, with approximately 95% disappearing along a 1 km transect. In the treatment forest, floodwater NO ₃ ^– concentrations decreased from 1000 M at the inflow point to 50 M along the 1 km transect. Nitrous oxide concentrations increased from 0.25 M to 1.2 M within the first 100 m, but decreased to 0.1 M over the next 900 m. The initial increase in N₂O was presumably a result ofin situ denitrification. Model analyses indicated that denitrification was directly associated with nitrification and was limited by the availability of NO ₃ ^– produced by nitrification. Due to different redox potential optima, coupling of nitrification and denitrification was a function of a balance of environmental conditions that was moderately favorable to both processes. N removal efficiency was largely dependent on the proportion of effluent NH ₄ ⁺ to NO ₃ ^– . When NH ₄ ⁺ /NO ₃ ^– was 1, average N removal efficiency ranged from 95–100%, but ratios that were >1 reduced average efficiencies to as low as 57%. Actual effluent NH ₄ ⁺ /NO ₃ ^– loading ratios at this site are approximately 0.2 and are consistently <1.     

Nitrate and phosphate removal by<Emphasis Type="Italic"> Spirulina platensis</Emphasis>

The cyanobacterium Spirulina platensis was used to verify the possibility of employing microalgal biomass to reduce the contents of nitrate and phosphate in wastewaters. Batch tests were carried out in 0.5 dm³ Erlenmeyer flasks under conditions of light limitation (40 mol quanta m^–2 s^–1) at a starting biomass level of 0.50 g/dm³ and varying temperature in the range 23–40°C. In this way, the best temperature for the growth of this microalga (30°C) was determined and the related thermodynamic parameters were estimated. All removed nitrate was used for biomass growth (biotic removal), whereas phosphate appeared to be removed mainly by chemical precipitation (abiotic removal). The best results in terms of specific and volumetric growth rates ( =0.044 day^–1, Q _x =33.2 mg dm^–3 day^–1) as well as volumetric rate and final yield of nitrogen removal ( =3.26 mg dm^–3 day^–1, =0.739) were obtained at 30°C, whereas phosphorus was more effectively removed at a lower temperature. In order to simulate full-scale studies, batch tests of nitrate and phosphate removal were also performed in 5.0 dm³ vessels (mini-ponds) at the optimum temperature (30°C) but increasing the photon fluence rate to 80 mol quanta m^–2 s^–1 and varying the initial biomass concentration from 0.25 to 0.86 g/dm³. These additional tests demonstrated that an increase in the inoculum level up to 0.75 g/dm³ enhanced both NO₃ ^– and PO₄ ^3– removal, confirming a strict dependence of these processes on biomass activity. In addition, the larger surface area of the ponds and the higher light intensity improved removal yields and kinetics compared to the flasks, particularly concerning phosphorus removal ( =0.032–0.050 day^–1, Q _x =34.7–42.4 mg dm^–3 day^–1, =3.24–4.06 mg dm^–3 day^–1, =0.750–0.879, =0.312–0.623 mg dm^–3 day^–1, and =0.224–0.440).     

Nitrate deposition in northern hardwood forests and the nitrogen metabolism of Acer saccharum marsh

It is generally assumed that plant assimilation constitutes the major sink for anthropogenic Nitrate NO ₃ ^– deposited in temperate forests because plant growth is usually limited by nitrogen (N) availability. Nevertheless, plants are known to vary widely in their capacity for NO ₃ ^– uptake and assimilation, and few studies have directly measured these parameters for overstory trees. Using a combination of field and greenhouse experiments, we studied the N nutrition of Acer saccharum Marsh. in four northern hardwood forests receiving experimental NO ₃ ^– additions equivalent to 30 kg N ha^–1 year^–1. We measured leaf and fine-root nitrate reductase activity (NRA) of overstory trees using an in vivo assay and used ¹⁵N to determine the kinetic parameters of NO ₃ ^– uptake by excised fine roots. In two greenhouse experiments, we measured leaf and root NRA in A. saccharum seedlings fertilized with 0–3.5 g NO ₃ ^– –N m^–2 and determined the kinetic parameters of NO ₃ ^– and NH ₄ ⁺ uptake in excised roots of seedlings. In both overstory trees and seedlings, rates of leaf and fine root NRA were substantially lower than previously reported rates for most woody plants and showed no response to NO ₃ ^– fertilization (range = non-detectable to 33 nmol NO ₂ ^– g^–1 h^–1). Maximal rates of NO ₃ ^– uptake in overstory trees also were low, ranging from 0.2 to 1.0 mol g^–1 h^–1. In seedlings, the mean V _max for NO ₃ ^– uptake in fine roots (1 mol g^–1 h^–1) was approximately 30 times lower than the V _max for NH ₄ ⁺ uptake (33 mol g^–1 h^–1). Our results suggest that A. saccharum satisfies its N demand through rapid NH ₄ ⁺ uptake and may have a limited capacity to serve as a direct sink for atmospheric additions of NO ₃ ^– .     

Nitrogen fixation by periphyton and plankton on the Amazon floodplain at Lake Calado

Nitrogen fixation by periphyton and plankton was measured on the Amazon flood-plain using the acetylene reduction method calibrated with¹⁵N-N₂. The average ratio (± SD) of moles C₂H₄ reduced per mole N₂-N fixed was 3.4 ± 0.7, similar to other studies. Periphyton and plankton had high rates of light-dependent nitrogen fixation, with dark nitrogen fixation averaging 26% of the average rates in the light. The average daily (24 h) rates for periphyton nitrogen fixation in 1989 and 1990 were 1.79 and 0.51 mmol N₂-N·m^–2·d^–1 respectively, which are comparable to summer rates in many temperate cyanobacterial assemblages. Nitrogen fixation was depressed at N0₃ ^– concentrations as low as 0.5 M, and was below detection limits at concentrations of 4 M, which occurred during periods of river flooding. Planktonic nitrogen fixation rates were high (0.5–0.8 mmol N₂-N·m^–2·d^–1) during the high-water and drainage phases of the annual hydrograph when the floodplain waters were draining towards the river (low NO₃ ^–), but rates were undetectable (< 0.05 mmol N₂-N·m^–2·d^–1) when there was river flooding (high NO₃ ^–). Nitrogen fixation by periphyton and plankton in 1989–1990 accounted for approximately 8% of previously reported total annual nitrogen inputs to the floodplain at Lake Calado.     

Ex Vitro Phenotype Stability is Affected by In Vitro Cultivation

Plant phenotype stability during ex vitro growth, one of the main requirements of plant micropropagation, was tested on tobacco. Plants cultivated in vitro in the presence of 3 % sucrose under photon flux density (PFD) of 200 mol m^–2 s^–1 (3 % HL plants) showed the best growth and photosynthetic parameters in the course of 7-day acclimation. However, significant change in phenotype of these plants appeared under a decrease in PFD to 50 mol m^–2 s^–1 during further ex vitro growth (in the period of 7^th – 17^th day). Much higher internodia elongation was found in 3 % HL plants in comparison with plants grown in vitro on sucrose media under PFD of 50 mol m^–2 s^–1 (3 % LL) or without sucrose either under PFD of 50 mol m^–2 s^–1 or 200 mol m^–2 s^–1 (0 % LL, 0 % HL). It can be presumed that 3 % HL plants show permanent demand for high PFD. Neither ABA or chlorophyll contents nor de novo thylakoid membrane synthesis were related to the morphogenic effect of low PFD. Changeable contents of hexoses in leaves of 3 % HL and 3 % LL plants were in no direct correlation to the elongated growth.     

Compartmentation and flux characteristics of nitrate in spruce

The radiotracer¹³N was used to undertake compartmental analyses for NO ₃ ^– in intact non-mycorrhizal roots ofPicea glauca (Moench) Voss. seedlings. Three compartments were defined, with half-lives of exchange of 2.5 s, 20 s, and 7 min. These were identified as representing surface adsorption, apparent free space, and cytoplasm, respectively. Influx, efflux, and net flux as well as cytoplasmic and apparent-free-space nitrate concentrations were estimated for three different concentration regimes of external nitrate. After exposure to external NO ₃ ^– for 3 d, influx was calculated to be 0.09 mol·g^–1·h^–1 (at 10 M [NO ₃ ^– ]_o), 0.5mol·g^–1·h^–1 (at 100 M [NO _inf3 ^sup– ]_o), and 1.2 mol · g^–1· h^–1 (at 1.5 mM [NO ₃ ^– ]_o). Efflux increased with increasing [NO ₃ ^– ]_o, constituting 4% of influx at 10 M, 6% at 100 M, and 21% at 1.5 mM. Cytoplasmic [NO ₃ ^– ] was estimated to be 0.3 mM at 10 uM [NO ₃ ^– ]_o, 2mM at 100 M [NO ₃ ^– ]_o, and 4mM at 1.5 mM [NO ₃ ^– ]_o, while free-space [NO ₃ ^– ] was 16 M, 173 M, and 2.2 mM, respectively. A series of experiments was carried out to confirm the identity of the compartments resolved by efflux analysis. Pretreatment at high temperature or application of 2-chloro-ethanol, sodium dodecyl sulphate or hydrogen peroxide made it possible to distinguish the metabolic (cytoplasmic) phase from the remaining two (physical) phases. Likewise, varying [Pi] of the medium altered efflux and thereby [NO ₃ ^– ]_cyt, but did not affect [NO ₃ ^– ]_{free space}.Abbreviations and Symbols [NO ₃ ^– ]_cyt cytoplasmic NO ₃ ^– concentration - [NO ₃ ^– ]_{free space} apparent-free-space NO ₃ ^– concentration - [NO ₃ ^– ]_o concentration of NO ₃ ^– in the external solution - NO ₃ ^– flux - _co efflux from the cytoplasm - _oc influx to the cytoplasm - _net net flux - _xylem flux to the xylem - _red/vac combined flux to reduction and the vacuole The research was supported by a Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia Campus for providing¹³NO ₃ ^– , Drs. R.D. Guy and S. Silim for providing plant material, and Dr. M.Y. Wang, Mr. J. Mehroke and Mr. P. Poon for assistance in experiments and for helpful discussions.