Nitrogen and phosphorus uptake by the Brazilian kelp Laminaria abyssalis (Phaeophyta) in culture

The uptake of ammonium, nitrate and phosphate by laboratory-grown young sporophytes of Laminaria abyssalis was measured in a perturbed system (batch mode) at 18 °C and 35 ± 5 µE m^–2 s^–1 photon flux density. Uptake of all appeared to follow saturation-type nutrient uptake kinetics. The NO _inf3 ^sup– (K _s = 14.0 µM, V _max = 5.0 µmol h^–1 g^–1 dry wt) and NH _inf4 ^sup+ (K _s = 4.6 µM, V _max= 2.0 µmol h^–1 g^–1 dry wt) were taken up simultaneously, although NH _inf4 ^sup+ was taken up more rapidly. Values of K ₃ and V _max for phosphate were, respectively, 2.21 µM and 0.83 µmol h^–1 g^–1 dry wt. Nitrate and phosphate were both consumed in similar rates (V _max /Ks 0.37) at low concentrations. NH _inf4 ^sup+ , thus, might be a more efficient form of N fertilizer if artificial enrichment of seawater is used.     

Effects of temperature and CO2 enrichment on kinetic properties of NADP+-malate dehydrogenase in two ecotypes of Barnyard grass (Echinochloa crus-galli (L.) Beauv.) from contrasting climates

Summary The apparent energy of activation (E _a), Michaelis-Menten constant (K _mfor oxaloacetate), V _max/K _mratios and specific activities of NADP⁺-malate dehydrogenase (NADP⁺-MDH; EC 1.1.1.82) were analyzed in plants of Barnyard grass from Québec (QUE) and Mississippi (MISS) acclimated to two thermoperiods 28/22°C, 21/15°C, and grown under two CO₂ concentrations, 350 l l^-1 and 675 l l^-1. E _avalues of NADP⁺-MDH extracted from QUE plants were significantly lower than those of MISS plants. K _mvalues and V _max/K _mratios of the enzyme from both ecotypes were similar over the range of 10–30°C but reduced V _max/K _mratios were found for the enzyme of QUE plants at 30 and 40°C assays. MISS plants had higher enzyme activities when measured on a chlorophyll basis but this trend was reversed when activities were expressed per fresh weight leaf or per leaf surface area. Activities were significantly higher in plants of both populations acclimated to 22/28°C. CO₂ enrichment did not modify appreciably the catalytic properties of NADP⁺-MDH and did not have a compensatory effect upon catalysis or enzyme activity under cool acclimatory conditions. NADP⁺-MDH activities were always in excess of the amount required to support observed rates of CO₂ assimilation and these two parameters were significantly correlated. The enhanced photosynthetic performance of QUE plants under cold temperature conditions, as compared to that of MISS plants, cannot be attributed to kinetic differences of NADP⁺-malate dehydrogenase among these ecotypes.     

Kinetics of sulfate uptake by freshwater and marine species ofDesulfovibrio

Apparent half-saturation constants (K _m) and maximum uptake rates (V _max) for sulfate were determined in four species ofDesulfovibrio of freshwater and marine origin using a³⁵S-sulfate tracer technique. The lowerstK _m (5 M) was found in the freshwater speciesDesulfovibrio vulgaris (Marburg) and the highestK _m (77 M) in the marine speciesDesulfovibrio salexigens. Maximum specific rates of sulfate uptake (i.e.,V _max) were proportional to the growth rates observed in batch cultures. The halophilicDesulfovibrio salexigens did not change itsK _m andV _max between 1 and 6,000 M SO ₄ ^2- , and apparently did not induce a low-affinity uptake system at high sulfate concentrations. The low half-saturation constants measured for sulfate uptake explain why high rates of bacterial sulfate reduction occur in surface sediments of freshwater lakes, and why sulfate reduction can be a quantitatively important process in anaerobic carbon mineralization in low-sulfate environments. The results shows that extremely low sulfate concentrations must occur before sulfate reduction is completely outcompeted by methanogenesis.Abbreviations MPB methane producing bacteria - SRB sulfate reducing bacteria     

Evidence that the low-affinity K+ transport system of Rhodobacter capsulatus is a uniporter. The effects of ammonium on the transporter

Comparison of the rate of accumulation of ⁸⁶Rb⁺ by intact cells of Rhodobacter capsulatus during short periods of illumination with the Rb⁺-dependent membrane ionic current measured by electrochromism supports the view that both activities reflect the operation of a low-affinity K⁺ transport system. In experiments performed under similar conditions the ratio of ⁸⁶Rb⁺ uptake to charge uptake was approx. 1.0, suggesting that the transport system operates as a uniporter. The addition of NH _inf4 ^sup+ to a cell suspension led to an increase in membrane ionic current but failed to inhibit the accumulation of ⁸⁶Rb⁺ during illumination. The presence of K⁺ and NH _inf4 ^sup+ inhibited the increase in cellular ATP levels at the onset of illumination. This effect was prevented by Cs⁺. The results are considered within the context of the hypothesis (Golby et al. Eur J Biochem 187: 589–597) that NH _inf4 ^sup+ can be transported by the K⁺ carrier and in the context of an alternative hypothesis that NH _inf4 ^sup+ increases the affinity of the K⁺ transport system for its natural substrate and for Rb⁺.Abbreviations pH pH difference across the cytoplasmic membrane - electrical potential difference across the cytoplasmic membrane     

Effect of NH4 +:NO3 - ratios on growth and nitrogen uptake by onions

The modelling of ion uptake by plants requires the measurement of kinetic and growth parameters under specific conditions. The objective of this study was to evaluate the effect of nine NH _inf4 ^sup+ :NO _inf3 ^sup− ratios on onions (Allium cepa L.). Twenty-eight to 84 day-old onion plants were treated with NH _inf4 ^sup+ :NO_f3/sup− ratios ranging from 0 to 100% of each ionic species in one mM solutions in a growth chamber. Maximum N influx (I_max) was assessed using the N depletion method. Except at an early stage, ionic species did not influence significantly I_max, the Michaelis constant (K_m) and the minimum concentration for net uptake (C_min). I_max for ammonium decreased from 101 to 59 pmole cm^-2 s^-1 while I_max for nitrate increased from 26 to 54 pmole cm^-2 s^-1 as the plant matured. On average, K_m and C_min values were 14.29 μM, and 5.06 μM for ammonium, and 11.90 μM and 4.54 μM for nitrate, respectively. In general, the effect of NH₄ ⁺:NO₃ ^- ratios on root weight, shoot weight and total weight depended on plant age. At an early stage, maximum plant growth and N uptake were obtained with ammonium as the sole source of N. At later stages, maximum plant growth and N uptake were obtained as the proportion of nitrate increased in the nutrient solution. The was no apparent nutrient deficiency whatever NH₄ ⁺:NO₃ ^- ratio was applied, although ammonium reduced the uptake of cations and increased the uptake of phosphorus. The research was supported by the Natural Sciences and Engineering Research Council of Canada.     

Ulva rigida (Ulvales,Chlorophyta) tank culture as biofilters for dissolved inorganic nitrogen from fishpond effluents

Ulva rigida was cultivated in 7501 tanks at different densities with direct and continuous inflow (at 2, 4, 8 and 12 volumes d^–1) of the effluents from a commercial marine fishpond (40 metric tonnes, Tm, of Sparus aurata, water exchange rate of 16 m³ Tm^–1) in order to assess the maximum and optimum dissolved inorganic nitrogen (DIN) uptake rate and the annual stability of the Ulva tank biofiltering system. Maximum yields (40 g DW m^–2 d^–1) were obtained at a density of 2.5 g FW 1^–1 and at a DIN inflow rate of 1.7 g DIN m^–2 d^–1. Maximum DIN uptake rates were obtained during summer (2.2 g DIN M^–2 d^–1), and minimum in winter (1.1 g DIN m^–2 d^–1) with a yearly average DIN uptake rate of 1.77 g DIN m^–2 d^–1 At yearly average DIN removal efficiency (2.0 g DIN m^–2 d^–1, if winter period is excluded), 153 m² of Ulva tank surface would be needed to recover 100% of the DIN produced by 1 Tm of fish.Abbreviations DIN= dissolved inorganic nitrogen (NH _inf4 ^sup+ + NO _inf3 ^sup– + NO _inf2 ^sup– ); - FW= fresh weight; - DW= dry weight; - PFD= photon flux density; - V= DIN uptake rate     

Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions

The interaction between ammonium and potassium during influx was examined in roots of dark-grown decapitated corn seedlings (Zea mays L., cv. Pioneer 3369A). Influx was measured during a 10-min exposure to either (¹⁵NH₄)₂SO₄ ranging from 10 to 200 M NH ₄ ⁺ with and without 200 M K(⁸⁶Rb)Cl or to K(⁸⁶Rb)Cl ranging from 10 to 200 M K⁺ with and without 200 M NH ₄ ⁺ as (¹⁵NH₄)₂SO₄. The simple Michaelis-Menten model described the data well only for potassium influx in the presence of ambient ammonium. For the other three instances, the data were improved by assuming that a second influx mechanism became operative as the low-concentration phase approached saturation. Two distinct mechanisms are thus indicated for both ammonium and potassium influx within the range of 10 to 200 M.The influx mechanism operating at low concentrations showed greater affinity for potassium than for ammonium, even though the capacity for ammonium transport was twice as large as that for potassium. It is suggested that this phase involved a common transport system for the two ions and that localized low acidity next to the internal surface, following H⁺ extrusion, favored ammonium deprotonation and dissociation from the transport system-ammonium complex. Parallel decreases in V _max and increases in K_m of the low-concentration saturable phase occurred for ammonium influx when ambient potassium was present and for potassium influx when ambient ammonium was present. The data support a mixed-type inhibition in each case. Simultaneous measurement of potassium and ammonium influx showed that they were highly negatively correlated at the lower concentrations, indicating that the extent to which influx of the inhibited ion was restricted was associated with influx of the inhibitor ion. Presence of ambient ammonium eliminated the second phase of potassium influx. In contrast, the presence of ambient potassium decreased the concentration at which the second phase of ammonium influx was initiated but did not restrict the rate.Paper no. 11131 of the Journal Series of the North Carolina Agricultural Research ServiceThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned     

The kinetics of NH4 + and NO3 − uptake by Douglas fir from single N-solutions and from solutions containing both NH4 + and NO3 −

The kinetics of NH₄ ⁺ and NO₃ ⁻ uptake in young Douglas fir trees (Pseudotsuga menziesii [Mirb.] Franco) were studied in solutions, containing either one or both N species. Using solutions containing a single N species, the V_max of NH₄ ⁺ uptake was higher than that of NO₃ ⁻ uptake. The K_m of NH₄ ⁺ uptake and K_m of NO₃ ⁻ uptake differed not significantly. When both NH₄ ⁺ and NO₃ ⁻ were present, the V_max for NH₄ ⁺ uptake became slightly higher, and the K_m for NH₄ ⁺ uptake remained in the same order. Under these conditions the NO₃ ⁻ uptake was almost totally inhibited over the whole range of concentrations used (10–1000 μM total N). This inhibition by NH₄ ⁺ occurred during the first two hours after addition. ei]{gnA C}{fnBorstlap}     

Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase

With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na⁺ + K⁺)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK _m is 2 orders of magnitude less and the V_max is two times greater, and dimethyl sulfoxide (Me₂SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK _m and V_max for K⁺-activated 3-OMFP hydrolysis as well as theK _0.5 for K⁺ activation. However, reducing the incubation pH increases inhibition by P_i and the V_max for 3-OMFP hydrolysis in the absence of K⁺. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K⁺-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K⁺, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na⁺, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na⁺ + K⁺)-phosphatase pathway, most apparent at suboptimal K⁺ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et₃N triethyl amine - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - MES 2-(N-morpholino)ethanesulfonate - Me₂SO dimethyl sulfoxide - NPP p-nitrophenyl phosphate - 3-OMFP 3-O-methylfluorescein phosphate - TNP-ATP 2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP     

Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K+ transport in a higher plant cell

Fusicoccin (FC) has long been known to promote K⁺ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H⁺ pumping stimulated in FC, could help drive K⁺ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V _m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K⁺ (⁸⁶Rb⁺) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K⁺ movement across the membrane could be modified in FC. We have explored a role for K⁺ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (⁸⁶Rb⁺) flux and in electrical current under voltage clamp was followed, using the K⁺ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K⁺-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K⁺ channel current contributed appreciably to charge balance maintainingV _m, and adding 10 mM TEA to block the current depolarized (positive-going)V _m; TEA also reduced⁸⁶Rb⁺ efflux by 68–80%. Following treatments with 10 M FC, both K⁺ channel current and⁸⁶Rb⁺ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced⁸⁶Rb⁺ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K⁺ (Rb⁺), despite membrane potentials which were 30–60 mVpositive of the K⁺ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K⁺ channels. They offer evidence of an energy-coupled mechanism for K⁺ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K⁺ transport capacity of the cells, independent of any changes in membrane potential.Abbreviations and symbols E _K equilibrium potential for K⁺ - FC fusicoccin - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - G _m membrane (slope) conductance atV _m - I-V current-voltage (relationship) - apparent rate constant for exchange - K _i ⁺ , K ₀ ⁺ intracellular, extracellular K⁺ (concentration) - TEA tetraethylammonium chloride - V _m free-running membrane potential (difference)     

Ammonium and nitrate as nitrogen sources in two Eriophorum species

Summary We compared ammonium and nitrate nutrition in Eriophorum scheuchzeri and E. vaginatum, two Alaskan sedges that are native to high- and low-fertility sites, respectively. When grown in solution culture, the two species were similar in their kinetics of NH _inf4 ^sup+ NO _inf3 ^sup- absorption: at nitrogen concentrations below 50 M, net NH _inf4 ^sup+ and NO _inf3 ^sup- were absorbed at similar rates, but at higher concentrations, net uptake of NO _inf3 ^sup- was significantly faster than that of NH _inf4 ^sup+ . The two species also showed similar abilities to assimilate NO _inf3 ^sup- . Growth of E. vaginatum under NO _inf3 ^sup- nutrition was only slightly less than that under NH _inf4 ^sup+ . The observed similarities between these species from contrasting edaphic habitats indicate that factors other than tissue-specific rates of nitrogen acquisition and assimilation may underlie local adaptation to soil N fertility. Moreover, the capacity of these species to exploit NO _inf3 ^sup- as a N source supports the view that NO _inf3 ^sup- availability may be significant even in wet, acidic, arctic soils.     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Uptake of iron byGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum (isolate 1–9) pathogenic on citrus fruits, appears to lack siderophore production. Iron uptake byG. candidum is mediated by two distinct iron-regulated, energy-and temperature-dependent transport systems that require sulfhydryl groups. One system exhibits specificity for either ferric or ferrous iron, whereas the other exhibits specificity for ferrioxamine-B-mediated iron uptake and presumably other hydroxamate siderophores. Radioactive iron uptake from⁵⁹FeCl₃ showed an optimum at pH 6 and 35° C, and Michaelis-Menten kinetics (apparentK _m = 3 m,V _max = 0.054 nmol · mg^–1 · min^–1). The maximal rate of Fe²⁺ uptake was higher than Fe³⁺ (V _max = 0.25 nmol · mg^–1 · min^–1) but theK _m was identical. Reduction of ferric to ferrous iron prior to transport could not be detected. The ferrioxamine B system exhibits an optimum at pH 6 and 40° C and saturation kinetics (K _m = 2 M,V _max = 0.22 nmol · mg^–1 · min^–1). The two systems were distinguished as two separate entities by negative reciprocal competition, and on the basis of differential response to temperature and phenazine methosulfate. Mössbauer studies revealed that cells fed with either⁵⁷FeCl₃ or⁵⁷FeCl₂ accumulated unknown ferric and ferrous binding metabolites.     

Non-mycorrhizal uptake of amino acids by roots of the alpine sedge Kobresia myosuroides: implications for the alpine nitrogen cycle

Non-mycorrhizal plants of the alpine sedge, Kobresia myosuroides, take up the amino acid glycine from nutrient solutions at greater rates than NO _inf3 ^sup- or NH _inf4 ^sup+ . The amino acids glutamate and proline were also taken up at high rates. Total plant biomass was twice as high after 4 months of growth on glycine, compared to NH₄NO₃, with significant increases in both root and leaf biomass. By taking advantage of differences in the ¹³C signature of air in the growth chamber and the glycine used for growth, a two-member mixing model was used to estimate that a significant amount of the glycine was taken up as intact molecules, enough to contribute 16% of the total carbon assimilation over a 4-month growing period. Glycine uptake was inhibited when roots were exposed to N₂ in place of air, and when the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added to the root solution. From these results it is concluded that glycine uptake occurs through active transport. Glycine uptake exhibited a Q₁₀ of 2.0 over the temperature range 5–15° C, with relatively high rates maintained at the lowest temperature measured (5° C). Roots of Kobreasia were not capable of taking up NH _inf4 ^sup+ at measureable rates. To our knowledge, this is the first report of a plant whose non-mycorrhizal roots cannot take up NH _inf4 ^sup+ . Measurements of three N fractions (NO _inf3 ^sup- , NH _inf4 ^sup+ , and total amino acids) in the soil pore water were made over two growing seasons in two Kobresia dry meadows using microlysimeters. At the West Knoll site, which is characterized by soils with average amounts of organic matter, the dominant forms of N in the soil pore water were NO _inf3 ^sup- and NH _inf4 ^sup+ (0–450 mol L^-1). Amino acid concentrations were generally less than 20 mol L^-1 at this site. At the East Knoll site, which is characterized by soils with higher than average amounts of organic matter, amino acids were generally present at higher concentrations (17–100 mol L^-1), compared to NO _inf3 ^sup- and NH _inf4 ^sup+ . The most abundant amino acids were glycine (10–100 mol L^-1), glutamate (5–70 mol L^-1), and late in the season cystein (5–15 mol L^-1). The results demonstrate that this sedge, which dominates dry meadow communities in many alpine ecosystems, is capable of taking up intact amino acids as a principal N source, and has access to high amino acid concentrations in certain alpine soils. Such uptake of organic N may accommodate plant N demands in the face of slow alpine N mineralization rates due to cold soil temperatures.     

Presence of an X AG − carrier in frog (Rana esculenta) red blood cells

Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K _m = 3 m and low capacity (V_max) 0.4 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X _AG ^– family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K _m 39 m but high capacity (V _max) 1.8 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1.     

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 ^– .     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Uptake of [14C]sucrose in isolated minor-vein networks of Commelina benghalensis L

Maceration with pectinase (4.5h) of Commelina benghalensis L. leaves stripped at either side yielded isolated vein networks consisting of four to five secondary veins and tertiary cross veins (=minor veins). Examination with Evans Blue and injection of Fluorescein F showed that 80% of the veins were viable. Proof of normal functioning of isolated minor veins was that [¹⁴C]sucrose fed to an apical vein network attached to the remaining intact part of the leaf was absorbed and finally arrived in the petiole. Sucrose uptake by veins obeyed Michaelis-Menten kinetics (K _m 5·10^-4 mol l^-1; V _max (light) 3.2 mol h^-1 g^-1 fresh weight, V _max (dark) 1.5 mol h^-1 g^-1 fresh weight). A linear component, not inhibited by carbonylcyanide m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid, was present. Maximal uptake took place at 5 mmol l^-1 K⁺; concentrations of K⁺ higher than 10 mmol l^-1 decreased the rate of uptake. The uptake rates by isolated veins and veins in situ (in disks) were in the same order of magnitude. Altogether, isolated veins promise to be a useful system for the study of loading.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediamine tetraacetic acid - PCMBS p-chloromercuribenzenesulfonic acid     

Ammonium uptake by Chlorella

The preincubation of Chlorella cells with glucose caused a tenfold increase of the maximal uptake rate of ammonium without change in the K _m (2 M). A similar stimulation of ammonium uptake was found when the cells were transferred to nitrogen-free growth medium. The time-course of uptake stimulation by glucose revealed a lag period of 10–20 min. The turnover of the ammonium transport system is characterized by a half-life time of 5–10 h, but in the presence of light 30% of uptake activity stayed even after 50 h. 6-Deoxyglucose was not able to increase the ammonium uptake rate. These data together were interpreted as evidence for induction of an ammonium transport system by a metabolite of glucose. Mechanistic studies of the ammonium transport system provided evidence for the electrogenic uptake of the ammonium ion. The charge compensation for NH ₄ ⁺ entry was achieved by immediate K⁺ efflux from the cells, and this was followed after 1 min by H⁺ extrusion. Ammonium accumulated in the cells; the rate of uptake was sensitive to p-trifluoromethoxy-carbonylcyanide-phenylhydrazon and insensitive to methionine-sulfoxime. Uptake studies with methylamine revealed that methylamine transport is obviously catalyzed by the ammonium transport system and, therefore, also increased in glucose-treated Chlorella cells.Abbreviation p.c. packed cells     

Transport and membrane binding of the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) in Xenopus laevis oocytes

Summary We have examined transport and membrane binding of 6-diazo-5-oxo-l-norleucine (DON, a photoactive diazo-analogue of glutamine) and their relationships to glutamine transport in Xenopus laevis oocytes. DON uptake was stereospecific and saturable (V _max of 0.44 pmol/oocyte · min and a K _m of 0.065 mm). DON uptake was largely Na^u+ dependent (80% at 50 m DON) and inhibited (>75%) by glutamine and arginine (substrates of the System B^0,+ transporter) at 1 mm. Glutamine and DON show mutual competitive inhibition of Na⁺-dependent transport. Preincubation of oocytes in medium containing 0.1 mm DON for 24 or 48 hr depressed the V _max for System B^0,+ transport (as measured by Na⁺-dependent glutamine uptake), this effect was highly specific (neither d-DON nor the System B^0,+ substrates glutamine and d-alanine showed any independent effect) and required Na⁺ ions. Glutamine (1 mm in preincubation medium) protected transport from inhibition by DON. The possibility that specific inactivation of System B^0,+ by DON reflects attachment of DON to the transporter was tested by examining the binding of [¹⁴C]DON to Xenopus oocyte membranes. Oocytes incubated in 100 mm NaCl in the presence of [¹⁴C]DON for up to 48 hr showed 2.4-fold higher ¹⁴C-binding to membranes than oocytes incubated in choline chloride. Na⁺-dependent DON binding (31 ± 11 fmol/g membrane protein) was suppressed by external glutamine, arginine or alanine and was largely confined to a membrane protein fraction of 48–65 kDa (as assessed by SDS-polyacrylamide gel electrophoresis). The present studies indicate that DON and glutamine uptake in oocytes are both mediated by System B^0,+ and demonstrate that DON binding to a particular membrane protein fraction is associated with inactivation of the transporter, offering the prospect of using [¹⁴C]DON as a covalent label for the transport protein in order to facilitate its isolation and subsequent biochemical characterization.This work was supported by The Wellcome Trust, Action Research for the Crippled Child, Ajinomoto GmbH, Pfrimmer GmbH, the Rank Prize Funds, the Medical Research Council and the University of Dundee. We are grateful to Dr. C.I. Pogson (Wellcome Research Laboratories) and Drs. J.C. Ellory and B. Elford (University of Oxford) for gifts of [¹⁴C]DON.