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.     

Nitrate Uptake into Barley (Hordeum vulgare) Plants : A New Approach Using ClO(3) as an Analog for NO(3)

Evidence is presented that chlorate is an extremely good analog for nitrate during nitrate uptake by intact barley (Hordeum vulgare cv. Fergus) roots. The depletion of ClO₃⁻ or NO₃⁻ from uptake media over 2 to 6 hours by seedlings was found to be dependent on combined NO₃⁻ plus ClO₃⁻ concentrations, and total anion uptake was equivalent at different NO₃⁻/ClO₃⁻ ratios. After loading barley seedlings with ³⁶ClO₃⁻ for 6 hours, kinetic parameters were derived from the analysis of efflux of [³⁶Cl] chlorate into unlabeled solution. On the basis of this analysis, the half times for exchange for the cytoplasmic and vacuolar phases were 17 minutes and 20 hours, respectively.     

Studies of the Uptake of Nitrate in Barley: I. Kinetics of NO(3) Influx

¹³NO₃⁻ was used to investigate patterns of NO₃⁻ influx into roots of barley plants (Hordeum vulgare L. cv Klondike) previously grown with (`induced') or without (`uninduced') a source of external NO₃⁻ ([NO₃⁻]₀). In both induced and uninduced plants, ¹³NO₃⁻ influx was biphasic in the range from 0.005 to 50 moles per cubic meter [NO₃⁻]₀. In the low concentration range (<1 mole per cubic meter for induced plants and <0.3 mole per cubic meter for uninduced plants), influx was saturable and V_max and K_m values for influx either increased or decreased according to NO₃⁻ pretreatment. By contrast, ¹³NO₃⁻ influx in the high concentration range revealed a strictly linear concentration dependence. These fluxes appeared to be mediated by a constitutive, rather than an inducible, transport system.     

Studies of the Regulation of Nitrate Influx by Barley Seedlings Using NO(3)

Using ¹³NO₃⁻, effects of various NO₃⁻ pretreatments upon NO₃⁻ influx were studied in intact roots of barley (Hordeum vulgare L. cv Klondike). Prior exposure of roots to NO₃⁻ increased NO₃⁻ influx and net NO₃⁻ uptake. This `induction' of NO<sub>3</sub><sup>−</sup> uptake was dependent both on time and external NO<sub>3</sub><sup>−</sup> concentration ([NO<sub>3</sub><sup>−</sup>]). During induction influx was positively correlated with root [NO<sub>3</sub><sup>−</sup>]. In the postinduction period, however, NO<sub>3</sub><sup>−</sup> influx declined as root [NO<sub>3</sub><sup>−</sup>] increased. It is suggested that induction and negative feedback regulation are independent processes: Induction appears to depend upon some critical cytoplasmic [NO<sub>3</sub><sup>−</sup>]; removal of external NO<sub>3</sub><sup>−</sup> caused a reduction of <sup>13</sup>NO<sub>3</sub><sup>−</sup> influx even though mean root [NO<sub>3</sub><sup>−</sup>] remained high. It is proposed that cytoplasmic [NO<sub>3</sub><sup>−</sup>] is depleted rapidly under these conditions resulting in `deinduction' of the NO₃⁻ transport system. Beyond 50 micromoles per gram [NO₃⁻], ¹³NO₃⁻ influx was negatively correlated with root [NO₃⁻]. However, it is unclear whether root [NO₃⁻] per se or some product(s) of NO₃⁻ assimilation are responsible for the negative feedback effects.     

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.     

Studies of the Uptake of Nitrate in Barley: III. COMPARTMENTATION OF NO3-

Compartmental analyses of intact roots of barley (Hordeum vulgareL. cv. Klondike) plants, grown with different levels of NO₃^–(up to 1·0 mol m^–3) in the external media, wereundertaken using ¹³NO₃^–. Two additional treatments, namelysodium dodecyl sulphate (SDS) or brief exposure to high temperature,designed to investigate the identity of the three NO₃^–compartments revealed by compartmental analyses, provided supportfor the identification of the latter as corresponding to superficialsolution, apoplasm, and cytoplasm. Half-lives for exchange ofthese compartments, 3 s, 30 s, and 7 mm, were unaffected bythe level of NO₃^– provided during growth. Independentestimates of ¹³NO₃^– fluxes obtained by direct methodsagreed well with values of fluxes calculated from the compartmentalanalyses. Cytoplasmic [NO₃^–], estimated from the compartmental analyses,were in the range from 1–37 mol m^–3, and increasedwith increasing [NO₃^–] of the medium. Such values forcytoplasmic [NO₃^–] are inconsistent with an earlier proposal(Siddiqi, Glass, Ruth, and Rufty, 1990; Glass, Siddiqi, Ruth,and Rufty, 1990) of passive NO₃^– uptake in the concentrationrange above 10 mol m^–3. A model, based upon localizeddistribution of nitrate reductase activity in epidermal cells,is proposed in which the proposed passive low affinity NO uptakeat high external [NO₃^–] is restricted to epidermal cells. During loading periods with ¹³NO₃^–, significant amountsof ¹³N were translocated to the shoot. Two pools of ¹³N, onebeing the root symplasm, appear to participate in the transferof labelled N to the shoot. Key words: Barley, compartmentation, nitrate, nitrate reductase, ¹³N     

Regulation of NO(3) Influx in Barley : Studies Using NO(3)

Short-term (10 minutes) measurements of plasmalemma NO₃⁻ influx (_oc) into roots of intact barley plants were obtained using ¹³NO₃⁻. In plants grown for 4 days at various NO₃⁻ levels (0.1, 0.2, 0.5 millimolar), _oc was found to be independent of the level of NO₃⁻ pretreatment. Similarly, pretreatment with Cl⁻ had no effect upon plasmalemma ¹³NO₃⁻ influx. Plants grown in the complete absence of ¹³NO₃⁻ (in CaSO₄ solutions) subsequently revealed influx values which were more than 50% lower than for plants grown in NO₃⁻. Based upon the documented effects of NO₃⁻ or Cl⁻ pretreatments on net uptake of NO₃⁻, these observations suggest that negative feedback from vacuolar NO₃⁻ and/or Cl⁻ acts at the tonoplast but not at the plasmalemma. When included in the influx medium, 0.5 millimolar Cl⁻ was without effect upon ¹³NO₃⁻ influx, but NH₄⁺ caused approximately 50% reduction of influx at this concentration.     

Studies of the Uptake of Nitrate in Barley : IV. Electrophysiology

Transmembrane electrical potential differences (Δψ) of epidermal and cortical cells were measured in intact roots of barley (Hordeum vulgare L. cv Klondike). The effects of exogenous NO₃⁻ on Δψ (in the concentration range from 100 micromolar to 20 millimolar) were investigated to probe the mechanisms of nitrate uptake by the high-affinity (HATS) and low-affinity (LATS) transport systems for NO₃⁻ uptake. Both transport systems caused depolarization of Δψ, demonstrating that the LATS (like the HATS) for NO₃⁻ uptake is probably mediated by an electrogenic cation (H⁺?) cotransport system. Membrane depolarization by the HATS was “inducible” by NO₃⁻, and saturable with respect to exogenous [NO₃⁻]. By contrast, depolarization by the LATS was constitutive, and first-order in response to external [NO₃⁻]. H⁺ fluxes, measured in 200 micromolar and in 5 millimolar Ca(NO₃)₂ solutions, failed to alkalinize external media as anticipated for a 2 H⁺:1 NO₃⁻ symport. However, switching from K₂SO₄ solutions (which were strongly acidifying) to KNO₃ solutions at the same K⁺ concentration caused marked reductions in H⁺ efflux. These observations are consistent with NO₃⁻ uptake by the HATS and the LATS via 2 H⁺:1 NO₃⁻ symports. These observations establish that the HATS for nitrate uptake by barley roots is essentially similar to those reported for Lemna and Zea mays by earlier workers. There are, nevertheless, distinct differences between barley and corn in their quantitative responses to external NO₃⁻.     

Studies of the Uptake of Nitrate in Barley : II. Energetics

Q₁₀ values for ¹³NO₃⁻ influx were determined in `uninduced' (NO<sub>3</sub><sup>−</sup>-starved) and `induced' (NO₃⁻-pretreated) roots of barley (Hordeum vulgare L.) plants at various concentrations of external NO₃⁻ ([NO₃⁻]₀). At 0.02 mole per cubic meter [NO₃⁻]₀, Q₁₀ values for influx were from 3 to 4 between 5 and 10°C. As [NO₃⁻]₀ increased Q₁₀ values decreased, reaching values of 1.2 and 2.0, respectively, at 20 moles per cubic meter in uninduced and induced plants. The metabolic dependence of ¹³NO₃⁻ influx at low and high [NO₃⁻]₀ (0.1 and 20.0 moles per cubic meter, respectively) in uninduced and induced plants was probed by the use of various inhibitors. These experiments confirmed the findings of the Q₁₀ studies, demonstrating that at low [NO₃⁻]₀¹³NO₃⁻ influx was extremely sensitive to metabolic inhibition. By contrast, at high [NO₃⁻]₀, influx was relatively insensitive to the presence of inhibitors.     

Sodium-Stimulated NO(3) Uptake in Amaranthus tricolor L. Plants

Nitrate uptake of Na⁺ -deficient Amaranthus tricolor L. cv Tricolor seedlings from complete culture solution was stimulated by about 210% within 5 hours by application of 0.5 millimolar NaCl. From a Na⁺ -preloading experiment, intracellular Na⁺ was shown to be responsible for the stimulation of NO₃⁻ uptake. The results suggest a possible role of Na⁺ in NO₃⁻ uptake in C₄ plants.     

Control of Net Uptake of Nutrients by Regulation of Influx in Barley Plants Recovering from Nutrient Deficiency

Rates of influx and net uptake of nitrate, phosphate and sulphatewere measured in intact barley plants, and concurrent effluxwas obtained by difference. Net uptake of these anions variedwidely depending on the nutrient status of the plants, and thedifferences in net uptake could be accounted for almost entirelyby changes in influx. Efflux played only a minor role in regulatingnet uptake of nitrate, phosphate or sulphate during recoveryfrom N-, P-, or S-deficiency. Nitrate influx and short-term ammonium absorption by N-deficientbarley plants were closely correlated, and varied in parallelwith rates of net uptake of nitrate or ammonium by similar plants.Again, it would seem that net uptake of ammonium is controlledpredominantly by changes in the rate of influx.Copyright 1993,1999 Academic Press Hordeum vulgare, barley, nutrient absorption, influx, nitrate, phosphate, sulphate, ammonium     

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-.     

Nitrate Inhibition of Legume Nodule Growth and Activity : II. Short Term Studies with High Nitrate Supply

Soybean plants (Glycine max [L.] Merr) were grown in sand culture with 2 millimolar nitrate for 37 days and then supplied with 15 millimolar nitrate for 7 days. Control plants received 2 millimolar nitrate and 13 millimolar chloride and, after the 7-day treatment period, all plants were supplied with nil nitrate. The temporary treatment with high nitrate inhibited nitrogenase (acetylene reduction) activity by 80% whether or not Rhizobium japonicum bacteroids had nitrate reductase (NR) activity. The pattern of nitrite accumulation in nodules formed by NR⁺ rhizobia was inversely related to the decrease and recovery of nitrogenase activity. However, nitrite concentration in nodules formed by NR⁻ rhizobia appeared to be too low to explain the inhibition of nitrogenase. Carbohydrate composition was similar in control nodules and nodules receiving 15 millimolar nitrate suggesting that the inhibition of nitrogenase by nitrate was not related to the availability of carbohydrate.

Nodules on plants treated with 15 millimolar nitrate contained higher concentrations of amino N and, especially, ureide N than control nodules and, after withdrawal of nitrate, reduced N content of treated and control nodules returned to similar levels. The accumulation of N₂ fixation products in nodules in response to high nitrate treatment was observed with three R. japonicum strains, two NR⁺ and one NR⁻. The high nitrate treatment did not affect the allantoate/allantoin ratio or the proportion of amino N or ureide N in bacteroids (4%) and cytosol (96%).

     

Nitrogen Isotope Fractionation Associated with Nitrate Reductase Activity and Uptake of NO(3) by Pearl Millet

Nitrogen isotope fractionation by Pearl Millet (Pennisetum americanum L. and P. mollissimum L.) grown on nitrate was associated with nitrate reductase activity. Fractionation was evidenced at the step of nitrate reduction when the substrate-to-enzyme ratio was high (possibly saturating for the active sites of the nitrate reductase enzyme), for instance in young seedlings having a low nitrate reductase activity or in seedlings grown on high nitrate concentration.     

Effect of Phloem-Translocated Malate on NO(3) Uptake by Roots of Intact Soybean Plants

In soybean (Glycine max L. Merr. cv Kingsoy), NO₃⁻ assimilation in leaves resulted in production and transport of malate to roots (B Touraine, N Grignon, C Grignon [1988] Plant Physiol 88: 605-612). This paper examines the significance of this phenomenon for the control of NO₃⁻ uptake by roots. The net NO₃⁻ uptake rate by roots of soybean plants was stimulated by the addition of K-malate to the external solution. It was decreased when phloem translocation was interrupted by hypocotyl girdling, and partially restored by malate addition to the medium, whereas glucose was ineffective. Introduction of K-malate into the transpiration stream using a split root system resulted in an enrichment of the phloem sap translocated back to the roots. This treatment resulted in an increase in both NO₃⁻ uptake and C excretion rates by roots. These results suggest that NO₃⁻ uptake by roots is dependent on the availability of shoot-borne, phloem-translocated malate. Shoot-to-root transport of malate stimulated NO₃⁻ uptake, and excretion of HCO₃⁻ ions was probably released by malate decarboxylation. NO₃⁻ uptake rate increased when the supply of NO₃⁻ to the shoot was increased, and decreased when the activity of nitrate reductase in the shoot was inhibited by WO₄²⁻. We conclude that in situ, NO₃⁻ reduction rate in the shoot may control NO₃⁻ uptake rate in the roots via the translocation rate of malate in the phloem.     

Growth and Nutrient Uptake by Barley (Hordeum vulgare L. cv Herta): Studies Using an N-(2-Hydroxyethyl)ethylenedinitrilotriacetic Acid-Buffered Nutrient Solution Technique (I. Zinc Ion Requirements)

The critical range of Zn2+ activity in nutrient solution required for optimum growth of barley (Hordeum vulgare L. cv Herta) was studied using the synthetic chelating agent N-(2-hydroxyethyl)ethylenedinitrilotriacetic acid to buffer micronutrient metal ions. The activity of Zn2+ was varied over a wide range from approximately 0.1 x 10-11 to 22 x 10-11 M Zn2+. The dry weight of barley shoots reached a maximum at Zn2+ activities above approximately 3 x 10-11 M and was clearly depressed when Zn2+ activities were below about 1 x 10-11 M. The relationship in shoots between dry weight and Zn concentrations supports the view that there is a critical Zn concentration of about 25 [mu]g g-1 dry weight in whole shoots of barley seedlings. When Zn2+ activities in solution were near or below approximately 3 x 10-11 M, barley shoots accumulated higher concentrations of P, Mn, Ca, Mg, and Na, whereas Cu concentrations were reduced. P and Mn began to accumulate in the shoots before differences in dry weights were apparent and provided the earliest index of Zn deficiency. In Zn-deficient roots, concentrations of Ca and Mg increased by 25 to 30%, and those of Fe and Mn more than doubled. Zn appears to play a special role in regulating uptake of several mineral nutrients in barley.     

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.     

Nitrate Utilization by the Diatom Skeletonema costatum: I. Kinetics of Nitrate Uptake

Nitrate uptake has been studied in nitrogen-deficient cells of the marine diatom Skeletonema costatum. When these cells are incubated in the presence of nitrate, this ion is quickly taken up from the medium, and nitrite is excreted by the cells. Nitrite is excreted following classical saturation kinetics, its rate being independent of nitrate concentration in the incubation medium for nitrate concentration values higher than 3 micromolar. Nitrate uptake shows mixed-transfer kinetics, which can be attributed to the simultaneous contributions of mediated and diffusion transfer. Cycloheximide and p-hydroxymercuribenzoate inhibit the carrier-mediated contribution to nitrate uptake, without affecting the diffusion component. When cells are preincubated with nitrate, the net nitrogen uptake is increased.     

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.     

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum: II. Effects of NO(3) and NH(4) Addition to CO(2) Efflux in the Light

The effects of nitrate and ammonium addition on net and gross photosynthesis, CO₂ efflux and the dissolved inorganic carbon compensation point of nitrogen-limited Selenastrum minutum Naeg. Collins (Chlorophyta) were studied. Cultures pulsed with nitrate or ammonium exhibited a marked decrease in both net and gross photosynthetic carbon fixation. During this period of suppression the specific activity of exogenous dissolved inorganic carbon decreased rapidly in comparison to control cells indicating an increase in the rate of CO₂ efflux in the light. The nitrate and ammmonium induced rates of CO₂ efflux were 31.0 and 33.8 micromoles CO₂ per milligram chlorophyll per hour, respectively, and represented 49 and 48% of the rate of gross photosynthesis. Nitrate addition to cells at dissolved inorganic carbon compensation point caused an increase in compensation point while ammonium had no effect. In the presence of the tricarboxylic acid cycle inhibitor fluoroacetate, the nitrate-induced change in compensation point was greatly reduced suggesting the source of this CO₂ was the tricarboxylic acid cycle. These results are consistent with the mechanism of N-induced photosynthetic suppression outlined by Elrifi and Turpin (1986 Plant Physiol 81: 273-279).