Ammonium Uptake by Rice Roots (II. Kinetics of 13NH4+ Influx across the Plasmalemma)

Short-term influxes of 13NH4+ were measured in intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings that were hydroponically grown at 2, 100, or 1000 [mu]M NH4+. Below 1 mM external concentration ([NH4+]0), influx was saturable and due to a high-affinity transport system (HATS). For the HATS, Vmax values were negatively correlated and Km values were positively correlated with NH4+ provision during growth and root [NH4+]. Between 1 and 40 mM [NH4+]0, 13NH4+ influx showed a linear response due to a low-affinity transport system (LATS). The 13NH4+ influxes by the HATS, and to a lesser extent the LATS, are energy-dependent processes. Selected metabolic inhibitors reduced influx of the HATS by 50 to 80%, but of the LATS by only 31 to 51%. Estimated values for Q10 (the ratio of rates at temperatures differing by 10[deg]C) for HATS were greater than 2.4 at root temperatures from 5 to 10[deg]C and were constant at approximately 1.5 between 5 and 30[deg]C for the LATS. Influx of 13NH4+ by the HATS was insensitive to external pH in the range from 4.5 to 9.0, but influx by the LATS declined significantly beyond pH 6.0. The data presented are discussed in the context of the kinetics, energy dependence, and the regulation of ammonium influx.     

Characterization and regulation of ammonium transport systems in Citrus plants

We have investigated both the kinetics and regulation of 15NH4+ influx in roots of 3-month-old hydroponically grown Citrus (Citrus sinensis L. Osbeck x Poncirus trifoliata Blanco) seedlings. The 15NH4+ influx is saturable below an external ammonium concentration of 1 mM, indicating the action of a high-affinity transport system (HATS). The HATS is under feedback repression by the N status of the plant, being down-regulated in plants adequately supplied with N during growth, and up-regulated by N-starvation. When assayed between 1 and 50 mM [15NH4+]0, the 15NH4+ influx showed a linear response typical of a low-affinity transport system (LATS). The activity of the LATS increased in plants supplied with NH4+ as compared with plants grown on an N-free medium. Transfer of the plants to N-free solution resulted in a marked decrease in the LATS-mediated 15NH4+ influx. Accordingly, resupply of NH4+ after N-starvation triggered a dramatic stimulation of the activity of the LATS. These data provide evidence that in Citrus plants, the LATS or at least one of its components is inducible by NH4+. Even when up-regulated, both the HATS and the LATS displayed a limited capacity, as compared with that usually found in herbaceous species. The use of various metabolic uncouplers or inhibitors indicated that 15NH4+ influx mediated by the HATS is strongly dependent on energy metabolism and H+ transmembrane electrochemical gradient. By contrast, the LATS is not affected by protonophores or inhibitors of the H(+)-ATPase, suggesting that its activity is mostly driven by the NH4+/NH3 transmembrane gradient. In agreement with these hypotheses, the HATS-mediated 15NH4+ influx was strongly inhibited when the solution pH was raised from 4 to 7, whereas influx mediated by the LATS was slightly stimulated.     

Potassium Fluxes in Chlamydomonas reinhardtii (I.Kinetics and Electrical Potentials)

Potassium influx and cellular [K+] were measured in the unicellular green alga Chlamydomonas reinhardtii after pretreatment in either 10 or 0 mM external K+ ([K]0). K+ (42K+ or 86Rb+) influx was mediated by a saturable, high-affinity transport system (HATS) at low [K+]0 and a linear, low-affinity transport system at high [K+]o. The HATS was typically more sensitive to metabolic inhibition (and darkness) than the low-affinity transport system. Membrane electrical potentials were determined by measuring the equilibrium distribution of tetraphenylphosphonium. These values, together with estimates of cytoplasmic [K+] (B. Malhotra and A.D.M. Glass [1995] Plant Physiol 108: 1537-1545), demonstrated that at 0.1 mM [K+]0 K+ uptake must be active. At higher [K+]0 (>0.3 mM) K+ influx appeared to be passive and possibly channel mediated. When cells were deprived of K+ for 24 h, the Vmax for the HATS increased from 50 x 10-6 to 85 x 10-6 nmol h-1 cell-1 and the Km value decreased from 0.25 to 0.162 mM. Meanwhile, cellular [K+] declined from 24 x 10-6 to 9 x 10-6 nmol cell-1. During this period influx increased exponentially, reaching its peak value after 18 h of K+ deprivation. This increase of K+ influx was not expressed when cells were exposed to inhibitors of protein synthesis. The use of 42K+ and 86Rb+ in parallel experiments demonstrated that Chlamydomonas discriminated in favor of K+ over Rb+, and this effect increased with the duration of K+ deprivation.     

Ammonium Uptake by Rice Roots (III. Electrophysiology)

The transmembrane electrical potential differences ([delta][psi]) were measured in epidermal and cortical cells of intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings grown in 2 or 100 [mu]M NH4+ (G2 or G100 plants, respectively). In modified Johnson's nutrient solution containing no nitrogen, [delta][psi] was in the range of -120 to -140 mV. Introducing NH4+ to the bathing medium caused a rapid depolarization. At the steady state, average [delta][psi] of G2 and G100 plants were -116 and -89 mV, respectively. This depolarization exhibited a biphasic response to external NH4+ concentration similar to that reported for 13NH4+ influx isotherms (M.Y. Wang, M.Y. Siddiqi, T.J. Ruth, A.D.M. Glass [1993] Plant Physiol 103: 1259-1267). Plots of membrane depolarization versus 13NH4+ influx were also biphasic, indicating distinct coupling processes for the two transport systems, with a breakpoint between two concentration ranges around 1 mM NH4+. The extent of depolarization was also influenced by nitrogen status, which was larger for G2 plants than for G100 plants. Depolarization of [delta][psi] due to NH4+ uptake was eliminated by a protonophore (carboxylcyanide-m-chlorophenylhydrazone), inhibitors of ATP synthesis (sodium cyanide plus salicylhydroxamic acid), or an ATPase inhibitor (diethylstilbestrol). The results of these observations are discussed in the context of the mechanisms of NH4+ uptake by high- and low-affinity transport systems operating across the plasma membranes of root cells.     

Kinetics of NO3- Influx in Spruce

Influxes of 13NO3- across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Three kinetically distinct uptake systems for NO3- were identified. In seedlings not previously exposed to external NO3-, a single Michaelis-Menten-type constitutive high-affinity transport system (CHATS) operated in a 2.5 to 500 [mu]M range of external NO3- [NO3-]o. The Vmax of this system was 0.1 [mu]mol g-1 h-1, and the Km was approximately 15 [mu]M. Following exposure to NO3- for 3 d, this CHATS activity was increased approximately 3-fold, with no change of Km. In addition, a NO3--inducible high-affinity system became apparent with a Km of approximately 100[mu]M. The combined Vmax for these discrete saturable components was 0.7 [mu]mol g-1 h-1. In both uninduced and induced plants a linear low-affinity system, additive to CHATS and an NO3--inducible high-affinity system, operated at [NO3-]o [greater than or equal to] 1 mM. The time taken to achieve maximal rates of uptake (full induction) was 2 d from 1.5 mM [NO3-]o and 3 d from 200 [mu]M [NO3-]o.     

Kinetics of NH 4 + uptake by the arbuscular mycorrhizal fungus Rhizophagus irregularis

The kinetics and energetics of (15)NH (4) (+) uptake by the extraradical mycelium of the arbuscular mycorrhizal fungus Rhizophagus irregularis were investigated. (15)NH (4) (+) uptake increased with increasing substrate concentration over the concentration range of 0.002 to 25?mM. Eadie-Hofstee plots showed that ammonium (NH (4) (+) ) uptake over this range was biphasic. At concentrations below 100?μM, NH (4) (+) uptake fits a Michaelis-Menten curve, typical of the activity of a saturable high-affinity transport system (HATS). At concentrations above 1?mM, NH (4) (+) influx showed a linear response typical of a nonsaturable low-affinity transport system (LATS). Both transport systems were dependent on external pH. The HATS and, to a lesser extent, the LATS were inhibited by the ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP) and the ATP-synthesis inhibitor 2,4-dinitrophenol. These data indicate that the two NH (4) (+) transport systems of R. irregularis are dependent on metabolic energy and on the electrochemical H(+) gradient. The HATS- and the LATS-mediated (15)NH (4) (+) influxes were also regulated by acetate. This first report of the existence of active high- and low-affinity NH4(+) transport systems in the extraradical mycelium of an arbuscular mycorrhizal fungus and provides novel information on the mechanisms underlying mycosymbiont uptake of nitrogen from the soil environment.     

Ammonium Uptake by Rice Roots (I. Fluxes and Subcellular Distribution of 13NH4+)

The time course of 13NH4+ uptake and the distribution of 13NH4+ among plant parts and subcellular compartments was determined for 3-week-old rice (Oryza sativa L. cv M202) plants grown hydroponically in modified Johnson's nutrient solution containing 2,100, or 1000 [mu]M NH4+ (referred to hereafter as G2, G100, or G1000 plants, respectively). At steady state, the influx of 13NH4+ was determined to be 1.31, 5.78, and 10.11 [mu]mol g-1 fresh weight h-1, respectively, for G2, G100, and G1000 plants; efflux was 11, 20, and 29%, respectively, of influx. The NH4+ flux to the vacuole was calculated to be between 1 and 1.4 [mu]mol g-1 fresh weight h-1. By means of 13NH4+ efflux analysis, three kinetically distinct phases (superficial, cell wall, and cytoplasm) were identified, with t1/2 for 13NH4+ exchange of approximately 3 s and 1 and 8 min, respectively. Cytoplasmic [NH4+] was estimated to be 3.72, 20.55, and 38.08 mM for G2, G100, and G1000 plants, respectively. These concentrations were higher than vacuolar [NH4+], yet 72 to 92% of total root NH4+ was located in the vacuole. Distributions of newly absorbed 13NH4+ between plant parts and among the compartments were also examined. During a 30-min period G100 plants metabolized 19% of the influxed 13NH4+. The remainder (81%) was partitioned among the vacuole (20%), cytoplasm (41%), and efflux (20%). Of the metabolized 13N, roughly one-half was translocated to the shoots.     

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.     

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.     

Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium.

A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.     

Modulation by external Ca2+ and nicardipine of Ca2+ influx and cytosolic concentration in human erythrocytes

Variations of Ca2+ influx (evaluated by the initial rate of 45Ca2+ uptake) and cytosolic free Ca2+ concentration ([Ca2+]i, measured with fura-2) were investigated in human erythrocytes. When external Ca2+ concentration ([Ca2+]o) rose from 1 to 2 mM, the initial rate of Ca2+ influx nearly doubled whereas [Ca2+]i increased only by 15%. Nicardipine dose-dependently decreased both initial rate of Ca2+ influx and [Ca2+]i (up to 53 and 18%. respectively at 10(-6) M). The less marked changes in [Ca2+]i than in Ca2+ influx indicate a partial adjustment of the Ca2+ extruding-pump activity to of Ca2+ influx. In vivo administration of nicardipine reduced [Ca2+]i only when its initial value exceeded 80 nM and prevented the rise in [Ca2+]i induced by the increase in [Ca2+]o. Our results indicate that nicardipine may reduce Ca2+ influx in human erythrocytes and participate in the control of [Ca2+]i when elevated.     

NO3- and ClO3- fluxes in the chl1-5 mutant of Arabidopsis thaliana. Does the CHL1-5 gene encode a low-affinity NO3- transporter?

The CHL1 gene is considered to encode a low-affinity transport system (LATS) for NO3- in Arabidopsis thaliana (Y.-F. Tsay, J.I. Schroeder, K.A. Feldmann, N.M. Crawford [1993] Cell 72: 705-713). However, the anticipated reduced NO3- uptake by the LATS associated with loss of CHL1 gene activity in chl1-5 deletion mutants was evident only when plants were grown on NH4NO3. When KNO3 was the sole N source, NO3- accumulation and short-term tracer influx (using 13NO3- and 15NO3-) in leaves and roots of wild-type and mutant plants were essentially identical. Nevertheless, root uptake of 36CIO3- by the LATS and CIO3- accumulation in roots and shoots of mutant plants were significantly lower than in wild-type plants when grown on KNO3. One explanation for these results is that a second LATS is able to compensate for the chl1-5 deficiency in KNO3-grown plants. Growth on NH4NO3 may down-regulate the second LATS enough that the anticipated difference in NO3- uptake becomes apparent.     

Major alterations of the regulation of root NO(3)(-) uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis

The role of AtNrt2.1 and AtNrt2.2 genes, encoding putative NO(3)(-) transporters in Arabidopsis, in the regulation of high-affinity NO(3)(-) uptake has been investigated in the atnrt2 mutant, where these two genes are deleted. Our initial analysis of the atnrt2 mutant (S. Filleur, M.F. Dorbe, M. Cerezo, M. Orsel, F. Granier, A. Gojon, F. Daniel-Vedele [2001] FEBS Lett 489: 220-224) demonstrated that root NO(3)(-) uptake is affected in this mutant due to the alteration of the high-affinity transport system (HATS), but not of the low-affinity transport system. In the present work, we show that the residual HATS activity in atnrt2 plants is not inducible by NO(3)(-), indicating that the mutant is more specifically impaired in the inducible component of the HATS. Thus, high-affinity NO(3)(-) uptake in this genotype is likely to be due to the constitutive HATS. Root (15)NO(3)(-) influx in the atnrt2 mutant is no more derepressed by nitrogen starvation or decrease in the external NO(3)(-) availability. Moreover, the mutant also lacks the usual compensatory up-regulation of NO(3)(-) uptake in NO(3)(-)-fed roots, in response to nitrogen deprivation of another portion of the root system. Finally, exogenous supply of NH(4)(+) in the nutrient solution fails to inhibit (15)NO(3)(-) influx in the mutant, whereas it strongly decreases that in the wild type. This is not explained by a reduced activity of NH(4)(+) uptake systems in the mutant. These results collectively indicate that AtNrt2.1 and/or AtNrt2.2 genes play a key role in the regulation of the high-affinity NO(3)(-) uptake, and in the adaptative responses of the plant to both spatial and temporal changes in nitrogen availability in the environment.     

Characterization of Paraquat Transport in Protoplasts from Maize (Zea mays L.) Suspension Cells

Protoplasts isolated from maize (Zea mays L.) suspension cells were used to study transport of paraquat. [14C]Paraquat uptake was measured in 400-[mu]L centrifuge tubes using silicon oil centrifugation techniques. Approximately 50% of accumulation from a 100 [mu]M paraquat solution occurred in the first 10 s, and net accumulation reached a maximum after about 10 min. Membrane binding accounted for about 30% of apparent accumulation. Concentration-dependent uptake kinetics were characterized by a non-saturating curve, which was resolved into a linear and a saturable component. The Km of the saturable component was 132 [mu]M, and the Vmax was 0.512 nmol [mu]L of protoplasts-1 min-1. In the absence of sucrose, the Vmax of the saturable component was reduced by 52%, suggesting that paraquat uptake across the plasmalemma is energy dependent. Measurement of concentration-dependent binding of paraquat to burst protoplasts showed a linear response. This suggests that the linear component from intact protoplast concentration kinetics represented paraquat binding to the plasmalemma surface. Calcium inhibited the saturable component, and this inhibition was shown by Lineweaver-Burk analysis to be noncompetitive. Putrescine, a divalent cationic polyamine with a charge distribution similar to that of paraquat, competitively inhibited paraquat uptake. These results show that paraquat transport characteristics at the plasmalemma of maize protoplasts are similar to those reported earlier for paraquat transport in roots of intact maize seedlings.     

Stimulation of Nitrate and Nitrite Efflux by Ammonium in Barley (Hordeum vulgare L.) Seedlings

The inhibitory effect of NH4+ on net NO3- uptake has been attributed to an enhancement of efflux and, recently, to an inhibition of influx. To study this controversy, we devised treatments to distinguish the effects of NH4+ on these two processes. Roots of intact barley (Hordeum vulgare L.) seedlings, uninduced or induced with NO3- or NO2-, were used. Net uptake and efflux, respectively, were determined by following the depletion and accumulation in the external solutions. In roots of both uninduced and NO2- -induced seedlings, NO3- efflux was negligible; hence, the initial uptake rates were equivalent to influx. Under these conditions, NH4+ had little effect on NO3- uptake (influx) rates by either the low- or high-Km uptake systems. In contrast, in plants preloaded with NO3-, NH4+ and its analog CH3NH3+ decreased net uptake, presumably by enhancing NO3- efflux. The stimulatory effect of NH4+ on NO3- efflux was a function of external NH4+ and internal NO3- concentration. These results were corroborated by the absence of any effect of NH4+ on NO2- uptake unless the roots were preloaded with NO2-. In this case NH4+ increased efflux and decreased net uptake. Hence, the main effect of NH4+ on net NO3- and NO2- uptake appears to be due to enhancement of efflux and not to inhibition of influx.     

Methylammonium as a Transport Analog for Ammonium in Tomato (Lycopersicon esculentum L.)

Methylammonium (CH3NH3+) has been widely used as an analog of ammonium (NH4+) for examining transport in bacteria and fungi. We compared the kinetics of root CH3NH3+ and NH4+ uptake from solution culture in intact tomato (Lycopersicon esculentum cv T5) plants. Efflux of NH4+ and CH3NH3+ was negligible. The apparent maximum rate of absorption (apparent Vmax) was similar for NH4+ and CH3NH3+, but the apparent affinity (apparent Km) was about 10-fold greater for NH4+ than for CH3NH3+. In characterizing the interaction between NH4+ and CH3NH3+ transport, we used [15N]NH4+ and [14C]CH3NH3+ as well as improved methods for analysis of nonisotopic CH3NH3+ and NH4+. CH3NH3+ acted as an inhibitor of NH4+ influx. Relatively low concentrations of NH4+ strongly inhibited CH3NH3+ influx. Treatments with 1 mM methionine sulfoximine that blocked NH4+ assimilation had little influence on NH4+ inhibition of CH3NH3+ influx. These results suggest that the two ions share a common transport system in tomato, but because this transport system has a much greater affinity for NH4+, CH3NH3+ may be used as a transport analog only when ambient concentrations of NH4+ are very low.     

Ammonia uptake and its effects on ionoregulation in the freshwater crayfish Pacifastacus leniusculus (Dana)

Exposure of adult crayfish Pacifastacus leniusculus to Artificial Freshwater (AFW) media containing 1.5 m and 0.15 mmol x l(-1) total ammonia [Tamm; 0.1 x acute lethal concentration (24 h LC50) and 0.01 x 24 h LC50] and adjusted to pH 6.5, pH 8.2 and pH 10.5 resulted in significant increases in haemolymph ammonia over a 24-h period. Ammonia accumulated most rapidly at pH 10.5. These media were chosen to expose animals to a range of different un-ionised ammonia (UIA) [NH3] and ionised ammonia [NH4+] concentrations. From comparisons of measured transepithelial potential differences (PDte) with calculated Nernst potentials (PDNH4+) for the known haemolymph-to-medium gradients of [NH4+], it was deduced that, in pH 8.2 and pH 6.5 AFW, NH4+ was not in thermodynamic equilibrium across the integument (presumably gill epithelium). In pH 10.5 AFW with 1.5 mmol x l(-1) Tamm (predominantly NH3), the accumulation of ammonia in the haemolymph was in the NH4+ form due to haemolymph pH regulation by the crayfish in this alkaline external medium. Measured net fluxes of ammonia (Jamm(net)) were inwardly directed and maximal when [NH3] was the main component externally, but were also significant at pH 8.2 with high [NH4+] ([NH4+]:[NH3] approximately 20:1). Haemolymph Na+ depletion was significant and, over the 24-h exposure period, most rapid in high [NH3] medium but [Cl-] was unaffected. However, paradoxically, sodium uptake (measured JNa(in) on immediate transfer to high Tamm medium) was not significantly inhibited when [NH3] was the predominant ammonia species. In 1.5 mmol x l(-1) Tamm (mainly [NH4+), VNa(in) (the active component of JNa(in)) was significantly inhibited, particularly at low external [Na+]. This inhibition could not be demonstrated as one of competition at an Na+/NH4+ apical gill exchange site. The resultant net efflux of sodium from the animal showed that the ability of the animals to balance sodium losses at low external [Na+] was severely affected. Longer exposure to pH 10.5 AFW with high [NH3] (12 h) resulted in significantly increased JNa(out), while not significantly affecting JNa(in). Analysis of urinary Na+ losses showed that, while urinary flow rate and water reabsorption was most likely unaffected by ammonia exposure, final urine [Na+] was significantly elevated. The resulting urinary Na+ loss accounted for 63% of the increased JNa(out) in high [NH3] medium.     

Investigation of the Apparent Induction of Nitrate Uptake in Barley (Hordeum vulgare L.) Using NO3--Selective Microelectrodes (Modulation of Coarse Regulation of NO3- Uptake by Exogenous Application of Downstream Metabolites in the NO3- Assimilatory Pathway)

The influence of a 12-h pretreatment with either NO3-, NH4+, glutamine, or glutamate (300 [mu]M) on the apparent induction of NO3- uptake was investigated. Net fluxes of NO3- into roots of intact, 7-d-old barley (Hordeum vulgare L. cv Prato) seedlings in solution culture were estimated from ion activity gradients measured with NO3--selective microelectrodes in the unstirred layer of solution immediately external to the root surface. Control plants, pretreated with nitrogen-free nutrient solution, exhibited a sigmoidal increase in net NO3- uptake, reaching a maximum rate between 8 and 9 h after first exposure to NO3-. Plants pretreated with NH4+ or Glu exhibited a delay of several hours in the initiation of the induction process after they had been exposed to NO3-. In Gln-pretreated plants, however, responses ranged from no delay of the induction process to delays comparable to those observed following NH4+ or Glu pretreatments. Only treatment with NO3-resulted in the induction of NO3- uptake, whereas pretreatments with NH4+, Gln, or Glu tended to delay induction of NO3- uptake upon subsequent exposure to NO3-.