Interactions between nitrate and ammonium uptake: variation with growth rate,nitrogen source and species

The interaction between nitrate and ammonium uptake was examined as a function of preconditioning growth rate and nitrogen source by adding nitrate, ammonium, or both to nitrogen-sufficient,-deficient, and-starvedSkeletonema costatum (Grev.) Cleve and nitrogen-deficientChaetoceros debilis Cleve. By simultaneously measuring the internal accumulation of intermediates of nitrogen assimilation and the rates of nitrogen assimilation, the metabolic control of nitrogen uptake could be assessed. After the simultaneous addition of nitrate and ammonium to culture, both nitrate and ammonium uptake rates were decreased in comparison with the rates observed when each was added alone, although nitrate uptake was usually decreased more than ammonium uptake. Since both nitrate and ammonium uptake rates vary with time, preconditioning growth conditions, nitrogen sources present, and species, it was necessary to use several different indices to quantify inhibition. In general, ammonium inhibition of nitrate uptake inS. costatum was greatest in cultures preconditioned to ammonium and those at low growth rates, whereas ammonium uptake was inhibited most in cultures preconditioned to nitrate. In nitrogen-deficientC. debilis, nitrate uptake was more inhibited by ammonium, but uptake returned to normal rates more quickly than inS. costatum, whereas inhibition of ammonium uptake was similar. These results explain why the interaction between nitrate and ammonium uptake in the field can be so variable. Inhibition of uptake is not controlled by internal ammonium or total amino acids, nor is it related to the inability to reduce nitrate. Instead, inhibition must be determined in part by the external concentration of nitrogen compounds and in part by some intermediate(s) of nitrogen assimilation present inside the cell.Bigelow Laboratory Contribution No. 82022     

Species differences in accumulation of nitrogen pools in phytoplankton

The changes in the intracellular concentrations of nitrate, ammonium, free amino acids, protein, DNA, RNA and total nitrogen were measured in batch cultures of seven species of marine phytoplankton as they progressed from being nitrogen sufficient to being nitrogen starved. After several days of nitrogen starvation, either nitrate or ammonium was added to the cultures, and the measurements were continued for 10 to 36 h. By this means it was possible to assess the long-term and short-term changes in cellular nitrogen compounds and how they relate to phytoplankton nitrogen uptake and growth. Considerable species differences were observed in the amounts and kinds of nitrogen compounds which were stored and the degree to which the utilization of these compounds could support growth if the external nitrogen supply is low or variable. Despite the species variation, the results suggest that the concentrations or ratios of a number of intracellular nitrogen compounds can be used to assess the nitrogen deficiency and/or growth rate of natural phytoplankton populations.Contribution No. 1373 from the School of Oceanography, University of Washington and Contribution No. 83006 from the Bigelow Laboratory for Ocean Sciences     

Effect of nitrogen supply on nitrogen uptake,accumulation and assimilation in Porphyra perforata (Rhodophyta)

Porphyra perforata J. Ag. was collected from a rocky land-fill site near Kitsilano Beach, Vancouver, British Columbia, Canada and was grown for 4 d in media with one of the following forms of inorganic nitrogen: NO ₃ ^- , NH ₄ ⁺ and NO ₃ ^- plus NH ₄ ⁺ and for 10 d in nitrogen-free media. Internal nitrogen accumulation (nitrate, ammonium, amino acids and soluble protein), nitrate and ammonium uptake rates, and nitrate reductase activity were measured daily. Short initial periods (10 to 20 min) of rapid ammonium uptake were common in nitrogen-deficient plants. In the case of nitrate uptake, initial uptake rates were low, increasing after 10 to 20 min. Ammonium inhibited nitrate uptake for only the first 10 to 20 min and then nitrate uptake rates were independent of ammonium concentration. Nitrogen starvation for 8 d overcame this initial suppression of nitrate uptake by ammonium. Nitrogen starvation also resulted in a decrease in soluble internal nitrate content and a transient increase in nitrate reductase activity. Little or no decrease was observed in internal ammonium, total amino acids and soluble protein. The cultures grown on nitrate only, maintained high ammonium uptake rates also. The rate of nitrate reduction may have limited the supply of nitrogen available for further assimilation. Internal nitrate concentrations were inversely correlated with nitrate uptake rates. Except for ammonium-grown cultures, internal total amino acids and soluble protein showed no correlation with uptake rates. Both internal pool concentrations and enzyme activities are required to interpret changes in uptake rate during growth.     

Nitrogen uptake kinetics in three year-classes of Laminaria groenlandica (Laminariales: Phaeophyta)

Nitrate and ammonium uptake rates were measured for three year-classes of the perennial macrophyte Laminaria groenlandica Rosenvinge, collected from nitrogen-depleted waters in Barkley Sound, British Columbia, Canada, in summer 1981. A time course of uptake rate revealed that ammonium uptake was high during the first hour and then decreased for all three year-classes; the opposite pattern was exhibited for the time course of nitrate uptake rate. Nitrate uptake rate increased linearly with nitrate concentration up to the highest level tested (60 M). The nitrate uptake rate of first-year plants was three times higher than second- and third-year plants; ammonium uptake rates showed similar patterns to those for nitrate. The interaction between nitrate and ammonium was examined for first-year plants. Nitrate and ammonium were taken up simultaneously and uptake rates were identical and equal to uptake rates when only nitrate or ammonium was present in the medium. Therefore, first-year plants are able to take up twice as much inorganic nitrogen per unit time when both nitrate and ammonium are present. First-year plants showed significant diel periodicity in ammonium uptake rates, whereas second- and third-year plants showed no periodicity in nitrate or ammonium uptake rates.     

Short-term interaction between nitrate and ammonium uptake inThalassiosira pseudonana: Effect of preconditioning nitrogen source and growth rate

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.     

Assessment of simultaneous uptake of nitrogenous nutrients (15N) and inorganic carbon (13C) by natural phytoplankton populations

The simultaneous uptake of nitrogenous nutrients and inorganic carbon was measured in shipboard incubations of natural phytoplankton populations, using tracer additions of ¹³C-bicarbonate and ¹⁵N-labelled nitrogenous substrates. From March 1991 through March 1992, three stations on the Scotian Shelf (eastern Canada) were sampled monthly at ten depths in the euphotic zone. Additions of labelled nitrogen compounds ranged between 0.5 and 98% of ambient concentrations. Most of the C/N (at/at) uptake ratios were lower than the Redfield ratio, suggesting that nitrogen was not limiting. The fixation of carbon with and without addition of nitrate, ammonium or urea was generally similar. Some samples presented significant differences in carbon uptake rate between the four treatments, but these differences were not related to nitrogen enrichment (percent or nitrogen species). Given these results, the double-labelling method appears to be a reliable tool for measuring the simultaneous uptake of carbon and nitrogen by natural phytoplankton.     

Enzyme activities in some marine phytoplankters and the effect of nitrogen limitation on nitrogen and carbon metabolism inChlorella stigmatophora

A survey of the distribution of six enzymes of nitrogen and carbohydrate metabolism was carried out with eleven species of marine phytoplankton representing seven classes. The activities of some enzymes, e.g. NADPH-glutamate dchydrogenase, were absent in several organisms. The effects of nitrogen deficiency on enzyme activities in nitrogen-limited chemostat cultures ofChlorella stigmatophora were investigated. Increasing nitrogen deficiency was accompanied by increases in the activity of nitrate reductase, NADPH-glutamate dehydrogenase and glutamine synthetase, and decreases in photosynthesis, respiration, chlorophyll and total cell protein. In nitrogen-deficient algae, an increase in the activity of the nitrogen-assimilating enzymes may facilitate the efficient utilization of available nitrogen under these conditions.Present address: School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0118, USA     

Development of the ability to take up L-lysine by the diatom Phaeodactylum tricornutum

Freshly harvested cells of Phaeodactylum tricornutum Bohlin grown with nitrate, ammonium or lysine as a sole nitrogen source had a low ability to take up lysine or arginine, but this ability increased when cells were deprived, over 48 h, of either nitrogen or carbon. The effects of nitrogen and carbon deprivation were additive, and the uptake ability was greatest in cells incubated in darkness in nitrogen-free medium. Uptake ability increased in cells illuminated in the presence of 10^-5 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) an inhibitor of photosynthetic electron transport. An inverse relationship between rate of development of the uptake system and rate of photosynthesis was also established. Development of the uptake system was prevented by cycloheximide or by anaerobiosis. Following transfer to a normal nitrate medium, illuminated cells lost the lysineuptake system by dilution as the cells grew. There was a linear and positive correlation between the initial rate of uptake of lysine and the maximum concentration which was maintained in the cells when equilibrium was reached, indicating that transinhibition of lysine uptake may occur and that the extent of this inhibition is related to the size of the internal amino acid pool. The relevance of the findings to the growth of phytoplankton in natural waters is discussed.     

Desiccation enhanced nitrogen uptake rates in intertidal seaweeds

Desiccation increased nitrate and ammonium uptake rates upon resubmergence in late summer populations of the intertidal macroalgae Gigartina papillata (C.Ag.) J.Ag., Enteromorpha intestinalis (L.) Grev., Fucus distichus L., and Pelvetiopsis limitata (Setch) Gardn. The ratio of nitrogen uptake rates in desiccated plants to rates in hydrated plants (controls) was correlated with the position of the species in the intertidal zone. Gracilaria pacifica Abbott., the species occurring at the lowest shore level, showed no enhancement of nitrogen uptake following desiccation. The high intertidal species such as P. limitata and F. distichus showed a two-fold enhancement of nitrate and ammonium uptake following more extensive desiccation (>30%) and continued uptake even following severe desiccation (50 to 60%). After the plants had been desiccated, the increase in nitrate uptake rates upon submergence lasted much longer than a similar enhancement of ammonium uptake. The duration of the enhanced nitrate uptake was similar to the time required for total rehydration but the uptake rates were not related to the state of rehydration. The potential contribution that this enhanced nitrogen uptake following desiccation could make to total nitrogen procurement for growth is discussed. The experiments were carried out in 1979 or 1980 and repeated in 1981.This paper is dedicated to Dr. R. F. Scagel on the occasion of his retirement for his outstanding contribution to phycology     

The rapid response of the marine diatom Skeletonema costatum to changes in external and internal nutrient concentration

A nitrogen-deficient batch culture of the marine diatom Skeletonema costatum, when resupplied with a mixture of nitrate and ammonium, showed an initial enhanced nitrate uptake rate leading to a large internal concentration (pool) of nitrate. Following this initial nitrate uptake event, nitrate uptake ceased, and nitrate assimilation was inhibited until the ammonium present was used. At this point, nitrate uptake resumed and nitrate assimilation began. No internal ammonium pool was observed during nitrate utilization, but a large nitrate pool remained throughout the utilization of external nitrate. The internal nitrate pool decreased rapidly after exhaustion of nitrate from the culture medium, but growth of cellular particulate nitrogen continued for about 24 h. A mathematical simulation model was developed from these data. The model cell consisted of a nitrate pool, ammonium pool, dissolved organic nitrogen pool, and particulate nitrogen. It was found that simple Michaelis-Menten functions for uptake and assimilation gave inadequate fit to the data. Michaelis-Menten functions were modified by inclusion of inhibitory and stimulatory feedback from the internal pools to more accurately represent the observed nutrient utilization.     

Seaweed diversity enhances nitrogen uptake via complementary use of nitrate and ammonium

The consequences of declining biodiversity remain controversial, in part because many studies focus on a single metric of ecosystem functioning and fail to consider diversity's integrated effects on multiple ecosystem functions. We used tide pool microcosms as a model system to show that different conclusions about the potential effects of producer diversity on ecosystem functioning may result when ecosystem functions are measured separately vs. together. Specifically, we found that in diverse seaweed assemblages, uptake of either nitrate or ammonium alone was equal to the average of the component monocultures. However, when nitrate and ammonium were available simultaneously, uptake by diverse assemblages was 22% greater than the monoculture average because different species were complementary in their use of different nitrogen forms. Our results suggest that when individual species have dominant effects on particular ecosystem processes (i.e., the sampling effect), multivariate complementarity can arise if different species dominate different processes. Further, these results suggest that similar mechanisms (complementary nutrient uptake) may underlie diversity-functioning relationships in both algal and vascular-plant-based systems.     

Interactions of inorganic nitrogen in the uptake and assimilation by marine phytoplankton

The effect of ambient ammonium concentration on the nitrate uptake rate of marine phytoplankton was investigated. These studies consisted of laboratory experiments using unialgal species and field experiments using natural phytoplankton communities. In laboratory experiments, ammonium suppressed the uptake rates of nitrate and nitrite. Approximately 30 min were required for ammonium to exhibit its fully inhibitory effect on nitrate uptake. At high ammonium concentration (>3 g-at/l), a residual nitrate uptake rate of approximately 0.006 h^-1 was observed. When the ambient ammonium concentration was reduced to a value less than 1 g-at/l, the suppressed nitrate uptake rate subsequently attained a value comparable to that observed before the addition of ammonium. A range of 25 to 60% reduction in the nitrate uptake rate of natural phytoplankton communities was observed at ambient ammonium concentrations of 1.0 g-at/l. A mechanism is proposed for the suppression of nitrate uptake rate by ammonium through feedback control of the nitrate permease system and/or the nitrate reductase enzyme system. The feedback control is postulated to be regulated by the level of total amino acids in the cell.Contribution No. 936 from the Department of Oceanography, University of Washington, Seattle, Washington 98195, USA. This paper represents a portion of a dissertation submitted to the Department of Oceanography, University of Washington, Seattle, in partial fulfillment of the requirements for the Ph.D. degree.     

Regulation of nitrate and ammonium uptake in the Greenland Sea

The uptake of nitrate and ammonium was investigated experimentally during early spring 1989 in the Greenland Sea, with particular attention placed on the roles of irradiance, nitrogen concentrations and nitrateammonium interactions. The phytoplankton assemblage was dominated by the colonial prymnesiophyte Phaeocystis pouchetii. Nitrate concentrations ranged from undetectable at the end of the cruise to greater than 10 M, and ammonium levels ranged from less than 0.1 to 1.9M. The uptake of both nitrate and ammonium as a function of irradiance was found to be a saturation response. Photoinhibition occurred and was found to be greater for ammonium uptake. Ammonium uptake also saturated at irradiance levels five times lower than those needed to saturate nitrate uptake. Nitrate and ammonium uptake as a function of nitrogen concentration also was characterized by a saturation response, with the estimated half-saturation constant (K _s) value for nitrate uptake being 0.29 M. Elevated ammonium concentrations inhibited nitrate uptake, and the response appeared to be one of exponential decrease with increasing concentrations of ammonium. The most important factor in the Greenland Sea influencing ammonium uptake during the spring was irradiace, while both irradiance and ammonium concentrations played major roles in regulating nitrate uptake and new production.     

Simultaneous nitrogen and silicate deficiency of a phytoplankton community in a coastal jet-front

The nutritional status of a phytoplankton community was investigated in a coastal jet-front located in the Gulf of St. Lawrence, Canada, in 1987. During the sampling period, the frontal community was mainly composed of the diatomsChaetoceros debilis, Skeletonema costatum, Thalassiosira gravida andC. pelagicus. As previously reported for the St. Lawrence, some frontal stations were depleted both in nitrate and silicate. At stations impoverished in nitrate, internal nitrate pool concentrations were low or undetectable, suggesting that cells had not, recently, been exposed to a nitrate flux which exceeded the nitrate assimilation rate. At these impoverished stations, however, ambient and intracellular concentrations of ammonium and urea were high, suggesting that the community was not nitrogen-deficient. The comparison between the ambient silicate concentrations and the silicate requirement (K _s ) of the dominant diatoms suggests thatC. debilis andS. costatum were Si-deficient. This is further supported by the low silicate uptake rates and intracellular concentrations measured at the silicate impoverished stations. The silicate deficiency also resulted in a decrease in the seston and phytoplankton N:C ratios.Please address all correspondence and requests for reprints to Dr Levasseur at his new address: Maurice Lamontagne Institute, 850 Route de la Mer, Mont-Joli, Québec G5H 3Z4, Canada     

Light-dependency of nitrate uptake by phytoplankton over the spring bloom in Auke Bay,Alaska

The effect of light intensity on nitrate uptake by natural populations of phytoplankton was examined by ¹⁵N traceruptake experiments during the spring (March–May 1987) in Auke Bay, Alaska. The data were fit to a rectangular hyperbolic model which included a term for dark uptake. Three types of curves described nitrate uptake as a function of light intensity. The first (Type I) had a low half-saturation light intensity (K _I), low chlorophyll-specific uptakes rates, no dark uptake and occasional photoinhibition. These were observed during a period of biomass decrease, accompanied by low daily light and strong wind, prior to the major bloom. The second type (Type II) had relatively high K _I, high chlorophyll-specific uptake rates, and no dark uptake. Type II curves were observed during most of the period prior to nitrate depletion in the surface waters. Types I and II both appeared prior to nitrate depletion in the water and reflected variations in the light history of the phytoplankton population. The third type (Type III) occurred in nitrate-deplete conditions, when nitrate uptake was less dependent on light intensity (i.e., high rates of dark uptake and lower K _I). Decreased light-dependency during this period was coupled with physiological nitrogen deficiency in the population. Comparing these parameters to those of photosynthetic carbon fixation, K _Ivalues of nitrate uptake were generally higher than those of photosynthesis prior to nitrate depletion, and lower during nutrient-deplete conditions.     

Nitrogen uptake and growth of the germlings and mature thalli of Fucus distichus

Fucus distichus L. was collected near Vancouver, Canada, in late fall and early winter, 1981. The effects of the forms of nitrogen (nitrate, ammonium or urea) and periodic exposure to air on growth, rhizoid development and nitrogen uptake in germlings was investigated. Gamete release, fertilization, germination and germling growth had no requirement for a specific form of nitrogen. Periodic exposure to air increased secondary rhizoid development twofold. Nitrate and ammonium uptake rates of the germlings were higher than for the mature thalli (20 to 40 times for nitrate and 8 times for ammonium), while the halfsaturation constant (K _s) values for nitrate were similar (1 to 5 M). The germlings showed saturable uptake kinetics but the mature thalli did not. When germlings were exposed to air it caused a 70% decrease in nitrate uptake, but not change in ammonium uptake. Ammonium uptake in the mature thalli was proportional to the ambient ammonium concentration. Nitrate uptake in the mature thalli appeared to follow saturation kinetics at low nitrate concentrations, but showed a non-saturable component at concentrations greater than 10 M. Presence of ammonium inhibited nitrate uptake by the mature plants but not by the germlings.     

不同氮肥对油菜B与Mo吸收的影响

利用盆栽试验研究了潮土中不同氮肥对油菜硼钼吸收的影响，结果表明，前期(5周)施氮并没有促进油菜的生长，而且大部分氮肥对油菜生长表现抑制作用，氯化铵处理的作用最大，硫酸铵和尿素处理次之，硝酸铵处理再次之；不同氮肥均有抑制油菜生长前期(5周)硼吸收和累积的作用，其中以氯化铵、硝酸铵和尿素的作用为甚；不同氮肥同样抑制油菜生长前期(5周)对钼的吸收和累积，以硫酸铵处理作用最突出，氯化铵处理次之。但随着时间的推移，10、15周不同氮肥大多促进油菜的生长，增加油菜对硼钼的吸收和累积，且各氮肥处理间的差异逐步减小。     

In-situ measurements of nitrogenous nutrient uptake by kelp (Ecklonia maxima) and phytoplankton in a nitrate-rich upwelling environment

Nitrogen uptake by the kelp Ecklonia maxima Osbeck and phytoplankton was examined under different conditions of nutrient availability in a kelp bed off the Cape of Good Hope by measuring nutrient depletion in large plastic bags by the kelp and ¹⁵N uptake by phytoplankton. E. maxima took up nitrate and ammonia, but not urea, and showed only a weak preference for reduced nitrogen. Phytoplankton absorbed all three forms of nitrogen available, with a preference for ammonia and urea. Ambient nitrate concentration exhibited a marked and rapid decrease with northerly winds and an increase in response to offshore southerly winds. Nitrogen uptake by E. maxima was linearly related to ambient concentration and did not saturate even at nitrate concentrations >20g-at N l^-1, resulting in a significantly higher tissue nitrogen content under upwelling conditions. Nitrate imported by upwelling was the chief source of nitrogen utilised within the kelp bed. Locally regenerated nitrogen (ammonia and urea) was calculated to contribute only ca 4% of total nitrogen uptake during upwelling and 30% during the relaxation or downwelling phase.     

Saturated uptake kinetics: transient response of the marine diatom Thalassiosira pseudonana to ammonium,nitrate, silicate or phosphate starvation

Changes in the saturated uptake kinetics of the limiting nutrient were followed as Thalassiosira pseudonana (Clone 3 H) batch cultures entered ammonium, nitrate, silicate and phosphate starvation. Cultures starved of ammonium or phosphate developed very high specific uptake capacities over a 24 to 48 h starvation period, due to both decreases in cell quota and increases in uptake rates per cell. In particular, the cell phosphorus quota decreased ca. 8-fold during phosphate starvation and specific uptake rates exceeded 100 d^-1. In contrast, cultures entering nitrate or silicate starvation underwent little or no further cell division, and the uptake capacity declined during starvation. After 24 to 48 h starvation, an induction requirement for uptake of nitrate or silicate was apparent. These responses are consistent with adaptation to the pattern of supply of these nutrients in the field.     

Characteristics of the uptake system for L-lysine and L-arginine inPhaeodactylum tricornutum

Cells ofPhaeodactylum tricornutum Bohlin develop the ability to take up L-lysine when they are deprived of nitrogen (illuminated in nitrogen-free medium), carbon (incubated in darkness) or both. Cells with a developed uptake system take up and accumulate lysine in an unchanged form. Uptake occurs under either aerobic or anaerobic conditions and is dependent on the presence of sodium⁺ ions (K _s ^{Na +}=,ca. 10 mM). Some potassium⁺ ions are necessary for uptake, presumably within the cells, but with potassium⁺-replete cells, increasing K⁺ concentration depresses lysine uptake. The lysine-uptake porter also transports L-arginine.K _s values are about 1.5 M for lysine and 0.5 M for arginine. It is, however, possible that the uptake system developed by incubating cells in darkness differs from that produced in light; it shows a pronounced pH optimum at pH 8.5, whereas the activity of the light-developed system declines from pH 6.5 to pH 9.0 and correlates well with the concentration of lysine⁺. The uptake system developed in darkness may also have a higher affinity for lysine. Lysine uptake is not inhibited by 1 mM concentrations of nitrate, nitrate, ammonium, or urea nor by similar concentrations of amphoteric or acidic amino acids.