Daily changes in nitrate influx,efflux and metabolism in maize and pearl millet

Maize (Zea mays L.) and pearl millet (Pennisetum americanum (L.) Leeke) seedlings were exposed to [¹⁵N]nitrate for 1-h periods at eight times during a 24-h period (16–8 h light-dark for maize; 14–10 h for millet). Influx of [¹⁵N]nitrate as well as its reduction and translocation were determined during each period. The efflux of previously absorbed [¹⁴N]nitrate to the uptake solution was also estimated. No marked diurnal changes in [¹⁴N]nitrate efflux or [¹⁵N]nitrate influx were evident in maize. In contrast, [¹⁴N]nitrate efflux from millet increased and eventually exceeded [¹⁵N]nitrate influx during the late dark and early light periods, resulting in net nitrate efflux from the roots. The dissimilarity of their diurnal patterns indicates that influx and efflux are independently regulated. In both species, [¹⁵N]nitrate reduction and ¹⁵N translocation to shoots were curtailed more by darkness than was [¹⁵N]nitrate influx. In the light, maize reduced 15% and millet 24% of the incoming [¹⁵N]nitrate. In darkness, reduction dropped to 11 and 17%, respectively. Since the accumulation of reduced-¹⁵N in shoots declined abruptly in darkness, whereas that in roots was little affected, it is suggested that in darkness [¹⁵N]nitrate reduction occurred primarily in roots. The decrease in nitrate uptake and reduction in darkness was not related to efflux, which remained constant in maize and did not respond immediately to darkness in pearl millet.Paper No. 6722 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh     

Exogenous NO(3) Influx and Endogenous NO(3) Efflux by Two Maize (Zea mays L.) Inbreds during Nitrogen Deprivation

The influence of nitrogen stress on net nitrate uptake resulting from concomitant ¹⁵NO₃⁻ influx and ¹⁴NO₃⁻ efflux was examined in two 12-day-old inbred lines of maize. Plants grown on ¹⁴NO₃⁻ were deprived of nitrogen for up to 72 hours prior to the 12th day and then exposed for 0.5 hour to 0.15 millimolar nitrate containing 98.7 atom% ¹⁵N. The nitrate concentration of the roots declined from approximately 100 to 5 micromolar per gram fresh weight during deprivation, and ¹⁴NO₃⁻ efflux was linearly related to root nitrate concentration. Influx of ¹⁵NO₃⁻ was suppressed in nitrogen-replete plants and increased with nitrogen deprivation up to 24 hours, indicating a dissipation of factors suppressing influx. Longer periods of nitrogen-deprivation resulted in a decline in ¹⁵NO₃⁻ influx from its maximal rate. The two inbreds differed significantly in the onset and extent of this decline, although their patterns during initial release from influx suppression were similar. Except for plants of high endogenous nitrogen status, net nitrate uptake was largely attributable to influx, and genetic variation in the regulation of this process is implied.     

Restricted nitrate influx and reduction in corn seedlings exposed to ammonium

The effect of ambient ammonium (0.5 millimolar [¹⁴NH₄]₂SO₄) added to a nutrient solution containing 1.0 millimolar K¹⁵NO₃, 99 atom per cent ¹⁵N, upon [¹⁵N]nitrate assimilation and utilization of previously accumulated [¹⁴N]nitrate was investigated. Corn seedlings, 5-day-old dark-grown decapitated (experiment I) and 10-day-old light-grown intact (experiment II), which had previously been grown on K¹⁴NO₃ nutrient solution, were used. In both experiments, the presence of ambient ammonium decreased [¹⁵N]nitrate influx (20% after 6 hours) without significantly affecting the efflux of previously accumulated [¹⁴N]nitrate. In experiment I, relative reduction of [¹⁵N]nitrate (reduction as a percentage of influx) was inhibited more than was [¹⁵N]nitrate influx. Nevertheless, in experiment I, where all reduction could be assigned to the root system, the absolute inhibition of reduction during the 12 hours (13 micromoles/root) was less than the absolute inhibition in influx (24 micromoles/root). The data suggest that the influence of ammonium on [¹⁵N]nitrate influx could not be totally accounted for by the decrease in the potential driving force which resulted from restricted reduction; an additional impact on the influx process is indicated. Reduction of [¹⁵N]nitrate in experiment II after 6 hours accounted for 30 and 18% of the tissue excess ¹⁵N in the control and ammonium treatments, respectively. Relative distribution of ¹⁵N between roots and exudate (experiment I), or between roots and shoots (experiment II) was not affected by ammonium. On the other hand, the accumulation of [¹⁵N]nitrate in roots, shoots, and xylem exudate was enhanced by ammonium treatment compared to the control, whereas the accumulation of reduced ¹⁵N was inhibited.     

Influx,efflux and net uptake of nitrate in Quercus suber seedlings

Nitrate influx, efflux and net nitrate uptake were measured for the slow-growing Quercus suber L. (cork-oak) to estimate the N-uptake efficiency of its seedlings when grown with free access to nitrate. We hypothesise that nitrate influx, an energetically costly process, is not very efficiently controlled so as to avoid losses through efflux, because Q. suber has relatively high respiratory costs for ion uptake. Q. suber seedlings were grown in a growth room in hydroponics with 1 mM NO₃ ^-. Seedlings were labelled with ¹⁵NO₃ ^- in nutrient solution for 5 min to measure influx and for 2 h for net uptake. Efflux was calculated as the difference between influx and net uptake. Measurements were made in the morning, afternoon and night. The site of nitrate reduction was estimated from the ratio of NO₃ ^- to amino acids in the xylem sap; the observed ratio indicated that nitrate reduction occurred predominantly in the roots. Nitrate influx was always much higher than net acquisition and both tended to be lower at night. High efflux occurred both during the day and at night, although the proportion of ¹⁵NO₃ ^- taken up that was loss through efflux was proportionally higher during the night. Efflux was a significant fraction of influx. We concluded that the acquisition system is energetically inefficient under the conditions tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relatively large nitrate efflux can account for the high specific respiratory costs for nitrate transport in slow-growing grass species

In this paper we address the question why slow-growing grass species appear to take up nitrate with greater respiratory costs than do fast-growing grasses when all plants are grown with free access to nutrients. Specific costs for nitrate transport, expressed as moles of ATP per net mole of nitrate taken up, were 1.5 to 4 times higher in slow-growing grasses than in fast-growing ones (Scheurwater et al., 1998, Plant, Cell & Environ. 21, 995–1005). The net rate of nitrate uptake is determined by two opposing nitrate fluxes across the plasma membrane: influx and efflux. To test whether differences in specific costs for nitrate transport are due to differences in the ratio of nitrate influx to net rate of nitrate uptake, nitrate influx and the net rate of nitrate uptake were measured in the roots of two fast-growing ( Dactylis glomerata L. and Holcus lanatus L.) and two slow-growing (Deschampsia flexuosa L. and Festuca ovina L.) grass species at four points during the diurnal cycle, using ¹⁵NO₃ ^-. Efflux was calculated by subtraction of net uptake from influx; it was assumed that efflux of nitrogen represents the flux of nitrate. Transfer of the plants to the solution containing the labelled nitrate did not significantly affect nitrate uptake in the present grass species. The net rate of nitrate uptake was highest during the middle of the light period in all species. Diurnal variation in the net rate of nitrate uptake was mostly due to variation in nitrate influx. Variation in nitrate efflux did not occur in all species, but efflux per net mole of nitrate taken up was higher during darkness than in the light in the slow-growing grasses. The two fast-growing species, however, did not show diurnal variation in the ratio of efflux to net nitrate uptake. Integrated over 24 hours, the slow-growing grasses clearly exhibited higher ratios of influx to net uptake than the fast-growing grass species. Our results indicate that the higher ratio of nitrate influx to net nitrate uptake can account for higher specific costs for nitrate transport in slow-growing grass species compared with those in their fast-growing counterparts, possibly in combination with greater activity of the non-phosphorylating alternative respiratory path. Therefore, under our experimental conditions with plants grown at a non-limiting nitrate supply, nitrate uptake is less efficient (from the point of ATP consumption) in slow-growing grasses than in fast-growing grass species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of NaCl salinity on 15N-nitrate fluxes and specific root length in the halophyte Plantago maritima L.

The effect of salinity on nitrate influx, efflux, nitrate net uptake rate and net nitrogen translocation to the shoot was assessed in a ¹⁵N steady state labelling experiment in the halophyte Plantago maritima L. raised for 14 days on solution supplied with 50, 100 and 200 mol m^–3 sodium chloride or without sodium chloride. Additionally, salinity induced changes in root morphology were determined. Specific root length increased upon exposure to elevated sodium chloride concentrations due to variations in biomass allocation and length growth of the tap root. Changes in root morphology, however, had a minor effect on nitrate fluxes when expressed on a root fresh weight basis. The decreased rate of nitrate net uptake in plants grown on elevated levels of sodium chloride was almost entirely due to a decrease in nitrate influx. Expressed as a proportion of influx, nitrate efflux remained unchanged and was even lower at the highest salinity level. At all sodium chloride concentrations applied the initial rate of nitrogen net translocation to the shoot decreased relative to the rate of nitrate net uptake. It is concluded that under steady state conditions the negative effect of sodium chloride on the rate of nitrate net uptake at non growth-limiting salinity levels was due to the interaction between sodium chloride and nitrate transporters in the root plasma membrane and/or processes mediating the translocation of nitrogen compounds, possibly nitrate, to the shoot.     

Simultaneous Influx and Efflux of Nitrate during Uptake by Perennial Ryegrass

Experiments with intact plants of Lolium perenne previously grown with ¹⁴NO₃⁻ revealed significant efflux of this isotopic species when the plants were transferred to solutions of highly enriched ¹⁵NO₃⁻. The exuded ¹⁴NO₃⁻ was subsequently reabsorbed when the ambient solutions were not replaced. When they were frequently replaced, continual efflux of the ¹⁴NO₃⁻ was observed. Influx of ¹⁵NO₃⁻ was significantly greater than influx of ¹⁴NO₃⁻ from solutions of identical NO₃⁻ concentration. Transferring plants to ¹⁴NO₃⁻ solutions after a six-hour period in ¹⁵NO₃⁻ resulted in efflux of the latter. Presence of Mg²⁺, rather than Ca²⁺, in the ambient ¹⁵NO₃⁻ solution resulted in a decidedly increased rate of ¹⁴NO₃⁻ efflux and a slight but significant increase in ¹⁵NO₃⁻ influx. Accordingly, net NO₃⁻ influx was slightly depressed. A model in accordance with these observations is presented; its essential features include a passive bidirectional pathway, an active uptake mechanism, and a pathway for recycling of endogenous NO₃⁻ within unstirred layers from the passive pathway to the active uptake site.     

Influx and net flux of amino acids into larval and juvenile European flat oysters, Ostrea edulis (L.)

Influx and net flux of amino acids into veliger, pediveliger, and juvenile stages of the oyster, Ostrea edulus (L.), were examined. Influx of alanine and glycine was determined using ¹⁴C-labelled substrates and monitoring the disappearance of radioactivity in the medium. Net influx was determined fluorometrically by following the disappearance of primary amines from solutions containing known substrates. Rates of influx and net influx are comparable for juvenile Ostrea down to ambient substrate levels of 2 μM. Net influx of amino acids into veliger and pediveliger larvae occurs at all concentrations examined (9 μM minimum). Rates of maximum influx (J_maxⁱ) and ambient substrate concentration at which influx is half-maximal (K_t) are reported for each stage. The K_t for glycine and alamine influx is 35–40 μM, decreasing to ≈ 15 μM in 1.5 mm sized juvenile oysters.     

An Analysis of the Leakages of Sodium Ions into and Potassium Ions out of Striated Muscle Cells

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na]_i, and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na]_i and a component that increased linearly with increasing [Na]_i. Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na]_o, and a steady-state balance of the sodium ion fluxes occurred at [Na]_o = 40 mM. When solutions were K-free and contained 10^-4 M ouabain, net sodium influx varied linearly with [Na]_o and a steady state for the intracellular sodium was observed at [Na]_o = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na]_i but dependent on [Na]_o. Replacing external NaCl with MgCl₂ brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na]_o = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Some regulatory aspects of [14C]methylamine influx intoPisum sativum L. cv. Feltham First seedlings

[¹⁴C]Methylamine influx intoPisum sativum L. cv. Feltham First seedlings showed Michaelis-Menten-type kinetics with apparentV _max=49.2 mol·g^-1 FW·h^-1 and apparentK _m=0.51 mM. The competitive interactions between ammonium and methylamine were most obvious when biphasic kinetics were assumed with saturation of the first phase at 0.05 mM. The inhibitor constant for ammonium (K _i)=0.027 mM. When [¹⁴C]methylamine was used in trace amounts with ammonium added as substrate, the influx of tracer showed Michaelis-Menten-type kinetics with apparentV _max=3.46 mol·g^-1 FW·h^-1 and apparentK _m=0.15 mM. The initial rate of net ammonium uptake corresponded with that found when [¹⁴C]methylamine was used to trace ammonium influx. The latter was also stimulated by high pH_o and inhibited by nitrate. Ammonium pretreatment±methionine sulphoximine or glutamine pretreatment of the seedlings inhibited subsequent [¹⁴C]methylamine influx, while methylamine or asparagine pretreatment stimulated [¹⁴C]methylamine influx. There was also a stimulatory effect of prior inoculation withRhizobium. The results are discussed in terms of current models for the regulation of ammonium uptake in plants.     

Nitrogen-13 Studies of Nitrate Fluxes in Barley Roots: II. EFFECT OF PLANT N-STATUS ON THE KINETIC PARAMETERS OF NITRATE INFLUX

Influx of nitrate into the roots of intact barley plants wasfollowed over periods of 1–15 min using nitrogen-13 asa tracer. Based on measurements taken over 15 min from a rangeof external nitrate concentrations (0·2–250 mmolm^–3), the kinetic parameters of influx, I_max and K_m, werecalculated. Compared with plants grown in the presence of nitrate throughout,plants that had been starved of N for 3 d showed a significantlygreater value ofI_max for ¹³N-nitrate influx (by a factor of1·4–1·8), but a similar value of K_m (12–14mmol m^–3). Pre-treating N-starved plants with nitratefor about 5 h further increased the subsequent rate of ¹³N-nitrateinflux, but had little effect in the unstarved controls. Allowingfor this induction of additional nitrate transport, the differencein rates of nitrate influx in control and N-starved plants wassufficient to account for the previously-observed differencein net uptake by the two groups of plants. In barley plants grown without any exposure to nitrate, butwith ammonium as N-source, both I_max and K_m for subsequent ¹³N-nitrateinflux were significantly decreased (by about one-half) comparedwith the corresponding nitrate-grown controls. The importance of changes in the rate of influx in the regulationof net uptake of nitrate is discussed. Key words: Ion transport, nitrate, influx, kinetic parameters, N-deficiency     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): I. Control of Net Uptake by NO(3) Efflux

A computer-controlled multichannel data acquisition system was employed to obtain continuous measurements of net nitrate or chlorate uptake by roots of intact barley plants (Hordeum vulgare cv Betzes) using nitrate-specific electrodes. Plants, previously grown in solutions maintained at 10 or 200 micromolar NO₃⁻ (low N or high N conditions, respectively), were provided with 200 micromolar NO₃⁻ or ClO₃⁻ during the uptake period. Initial rates of NO₃⁻ uptake were several times higher in low N plants than in high N plants. Within 10 min, uptake in the former plants declined to a new steady rate which was sustained for the remainder of the experiment. No such time-dependent changes were evident in the high N plants. Rates and patterns of net chlorate uptake exhibited almost identical dependence upon previous nitrate provision. NO₃⁻ (³⁶ClO₃⁻) influx, by contrast, appeared to be independent of NO₃⁻ pretreatment prior to influx determination. Nitrate efflux, estimated by several different methods, was strongly correlated with internal nitrate concentration of the roots.     

Efficiency of nitrate uptake in spinach: impact of external nitrate concentration and relative growth rate on nitrate influx and efflux

Regulation of nitrate influx and efflux in spinach (Spinacia oleracea L., cv. Subito), was studied in short-term label experiments with 13N- and 15N-nitrate. Nitrate fluxes were examined in relation to the N demand for growth, defined as relative growth rate (RGR) times plant N concentration. Plants were grown at different nitrate concentrations (0.8 and 4 mM), with mineral composition of growth and uptake solutions identical. Nitrate influx, efflux and net nitrate uptake rate (NNUR) were independent of the external nitrate concentration, despite differences in internal nitrate concentration. At both N regimes, NNUR was adequate to meet the N demand for growth. RGR-related signals predominantly determined the nitrate fluxes. At high RGR (0.25 g g-1 day-1), nitrate influx was 20 to 40% lower and nitrate efflux was 50 to 70% lower than at lower RGR (0.17 g g-1 day-1); efflux:influx ratio (E:I) declined from 0.5 at low RGR to 0.2 at higher RGR. Thus, the efficiency of NNUR substantially increased with increasing RGR. Differences in nitrate translocation between morning and afternoon coincided with differences in nitrate efflux, which is in accordance with the suggested regulation of nitrate efflux by the root cytoplasmic nitrate concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

A substrate cycling model for nitrate uptake byPisum sativum seedlings: A key to sensitivity of response of net flux to substrate and effectors?

Summary Net nitrate uptake (J) intoPisum sativum L. seedlings has been investigated. J was high initially, but declined with time as NO ₃ ^– efflux (E) approached that of NO ₃ ^– influx (I). Both I and E were higher in plants which had been grown without N. J could be reversibly and immediately inhibited by 5 mmol m^–3 NH ₄ ⁺ , although plants grown in the presence of nitrate were less sensitive. A theoretical model which involves substrate cycling across the plasmalemma is shown to increase the sensitivity to substrate and effectors. It predicts that during growth of Pisum in N free media the cycling rate (E/I) is increased and the sensitivity of net flux of inhibition by NH ₄ ⁺ is highest. The model also provides a means for control of cytoplasmic nitrate pool size [NO ₃ ^– ] c.     

Effect of exogenous and endogenous nitrate concentration on nitrate utilization by dwarf bean

The effect of the exogenous and endogenous NO₃⁻ concentration on net uptake, influx, and efflux of NO₃⁻ and on nitrate reductase activity (NRA) in roots was studied in Phaseolus vulgaris L. cv. Witte Krombek. After exposure to NO₃⁻, an apparent induction period of about 6 hours occurred regardless of the exogenous NO₃⁻ level. A double reciprocal plot of the net uptake rate of induced plants versus exogenous NO₃⁻ concentration yielded four distinct phases, each with simple Michaelis-Menten kinetics, and separated by sharp breaks at about 45, 80, and 480 micromoles per cubic decimeter.

Influx was estimated as the accumulation of ¹⁵N after 1 hour exposure to ¹⁵NO₃⁻. The isotherms for influx and net uptake were similar and corresponded to those for alkali cations and Cl⁻. Efflux of NO₃⁻ was a constant proportion of net uptake during initial NO₃⁻ supply and increased with exogenous NO₃⁻ concentration. No efflux occurred to a NO₃⁻-free medium.

The net uptake rate was negatively correlated with the NO₃⁻ content of roots. Nitrate efflux, but not influx, was influenced by endogenous NO₃⁻. Variations between experiments, e.g. in NO₃⁻ status, affected the values of K_m and V_max in the various concentration phases. The concentrations at which phase transitions occurred, however, were constant both for influx and net uptake. The findings corroborate the contention that separate sites are responsible for uptake and transitions between phases.

Beyond 100 micromoles per cubic decimeter, root NRA was not affected by exogenous NO₃⁻ indicating that NO₃⁻ uptake was not coupled to root NRA, at least not at high concentrations.

     

Short Term Studies of Nitrate Uptake into Barley Plants Using Ion-Specific Electrodes and ClO(3): II. Regulation of NO(3) Efflux by NH(4)

The influence of NH₄⁺, in the external medium, on fluxes of NO₃⁻ and K⁺ were investigated using barley (Hordeum vulgare cv Betzes) plants. NH₄⁺ was without effect on NO₃⁻ (³⁶ClO₃⁻) influx whereas inhibition of net uptake appeared to be a function of previous NO₃⁻ provision. Plants grown at 10 micromolar NO₃⁻ were sensitive to external NH₄⁺ when uptake was measured in 100 micromolar NO₃⁻. By contrast, NO₃⁻ uptake (from 100 micromolar NO₃⁻) by plants previously grown at this concentration was not reduced by NH₄⁺ treatment. Plants pretreated for 2 days with 5 millimolar NO₃⁻ showed net efflux of NO₃⁻ when roots were transferred to 100 micromolar NO₃⁻. This efflux was stimulated in the presence of NH₄⁺. NH₄⁺ also stimulated NO₃⁻ efflux from plants pretreated with relatively low nitrate concentrations. It is proposed that short term effects on net uptake of NO₃⁻ occur via effects upon efflux. By contrast to the situation for NO₃⁻, net K⁺ uptake and influx of ³⁶Rb⁺-labeled K⁺ was inhibited by NH₄⁺ regardless of the nutrient history of the plants. Inhibition of net K⁺ uptake reached its maximum value within 2 minutes of NH₄⁺ addition. It is concluded that the latter ion exerts a direct effect upon K⁺ influx.     

The Kinetics of Chlorate Uptake by XD Tobacco Cells

The uptake of [³⁶Cl]chlorate by the 14U variant of the XD cell line of Nicotiana tobaccum L. cv Xanthi was investigated to examine the use of chlorate as a nitrate analog in transport studies. The kinetics of chlorate uptake against concentration was complex. Evidence was obtained, e.g., by means of nitrate competition, that these kinetics could be resolved into two components indicating the existence of two influx mechanisms: a saturable high affinity transport system (HATS) and a low affinity transport system (LATS) that showed first order kinetics. HATS has an apparent K_m for chlorate of 0.3 millimolar, and a marked pH dependence. The V_max dropped about fivefold as the pH was changed from the optimum pH (5.5-6.5), while the K_m remained virtually unchanged. The activity of HATS was completely inhibited by 15 millimolar nitrate and was less sensitive to chloride. LATS was inhibited by chloride and showed some inhibition by nitrate. It was concluded that [³⁶Cl]chlorate can be used as an analog for nitrate uptake studies only in a limited low concentration range where HATS is the main route for chlorate influx.     

Nitrogen Utilization in Lemna: II. Studies of Nitrate Uptake Using NO(3)

¹³N-labeled nitrate was used to trace short-term nitrate influx into Lemna gibba L. G3 in experiments where disappearance of both radioactivity and total nitrate from the incubation medium was measured continuously and simultaneously. In plants performing net nitrate uptake from an initial nitrate concentration of 40 to 60 micromolar, there was no discrepancy between net uptake and influx, irrespective of the N status of the plants, indicating that concomitant nitrate efflux was low or nil. Plants treated with tungstate to inactivate nitrate reductase were able to take up nitrate following induction of the uptake system by exposure to a low amount of nitrate. Also, in this case, net uptake was equivalent to influx. In tungstate-treated plants preloaded with nitrate, both net uptake and influx were nil. In contrast to these observations, a clear discrepancy between net uptake and influx was observed when the plants were incubated at an initial nitrate concentration of approximately 5 micromolar, where net uptake is low and eventually ceases. It is concluded that plasmalemma nitrate transport is essentially unidirectional in plants performing net uptake at a concentration of 40 to 60 micromolar, and that transport is nil when internal nitrate sinks (vacuole, metabolism) are eliminated. The efflux component becomes increasingly important when the external concentration approaches the threshold value for net nitrate uptake (the nitrate compensation point) where considerable exchange between internal and external nitrate occurs.     

Nitrogen Utilization in Lemna: II. Studies of Nitrate Uptake Using 13NO3?

¹³N-labeled nitrate was used to trace short-term nitrate influx into Lemna gibba L. G3 in experiments where disappearance of both radioactivity and total nitrate from the incubation medium was measured continuously and simultaneously. In plants performing net nitrate uptake from an initial nitrate concentration of 40 to 60 micromolar, there was no discrepancy between net uptake and influx, irrespective of the N status of the plants, indicating that concomitant nitrate efflux was low or nil. Plants treated with tungstate to inactivate nitrate reductase were able to take up nitrate following induction of the uptake system by exposure to a low amount of nitrate. Also, in this case, net uptake was equivalent to influx. In tungstate-treated plants preloaded with nitrate, both net uptake and influx were nil. In contrast to these observations, a clear discrepancy between net uptake and influx was observed when the plants were incubated at an initial nitrate concentration of approximately 5 micromolar, where net uptake is low and eventually ceases. It is concluded that plasmalemma nitrate transport is essentially unidirectional in plants performing net uptake at a concentration of 40 to 60 micromolar, and that transport is nil when internal nitrate sinks (vacuole, metabolism) are eliminated. The efflux component becomes increasingly important when the external concentration approaches the threshold value for net nitrate uptake (the nitrate compensation point) where considerable exchange between internal and external nitrate occurs.     

A comparison of regulatory effects of chloride on nitrate uptake, and of nitrate on chloride uptake into Pisum sativum seedlings

Net nitrate uptake, ³⁶ClO^?₃/NO^?₃ influx and ³⁶Cl^? influx into Pisum sativum L. cv. Feltham First seedlings have been examined following growth in culture medium containing different combinations of chloride and nitrate. When young (6 days old) seedlings, that had been grown in the absence of N were used, nitrate accumulation stimulated net nitrate uptake and ³⁶ClO^?₃/NO^?₃ influx (r²= 0.99) while chloride accumulation inhibited nitrate uptake and ³⁶ClO^?₃/NO^?₃ influx (r²= 0.65). When nitrate was provided during growth there was no effect of chloride pretreatment on net nitrate uptake and there was little effect of total [NO^?₃+ Cl^?]_i on ³⁶ClO^?₃/NO^?₃ influx (r²= 0.26). A direct effect of Cl^? on ³⁶ClO^?₃/NO^?₃ influx was only found when seedlings had been starved of N for more prolonged periods (14 days). When moderate chloride was supplied during growth, ³⁶Cl^? influx was insensitive to nitrate or chloride accumulated, but significantly correlated with log_e [NO^?₃+ Cl^?]_i (r²= 0.75). When trace amounts of Cl^? were supplied during growth ³⁶Cl^? influx was inhibited by (a) NO^?₃ in the external medium and (b) Cl^? pretreatment, but was insensitive to NO^?₃ pretreatment. The sensitivity of ³⁶Cl^? influx to external nitrate was not found following Cl^? pretreatment in the absence of nitrate. The possibility that there are two populations of chloride carriers which differ in their sensitivity to external nitrate is discussed. Tentative schematic models to account for the regulation of nitrate and chloride uptake are proposed in the context of current hypotheses for regulation of ion transport and control systems theory.