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.     

Effect of pH and calcium on short-term NO3- fluxes in roots of barley seedlings

The effect of pH and Ca2+ on net NO3- uptake, influx, and efflux by intact roots of barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with NO3- or NO2-. Net NO3- uptake and efflux, respectively, were determined by following its depletion from, and accumulation in, the external solution. Since roots of both uninduced and NO2(-)-induced seedlings contain little internal NO3- initial net uptake rates are equivalent to influx (M. Aslam, R.L. Travis, R.C. Huffaker [1994] Plant Physiol 106: 1293-1301). NO3-, uptake (influx) by these roots was little affected at acidic pH. In contrast, in NO3(-)-induced roots, which accumulate NO3-, net uptake rates decreased in response to acidic pH. Under these conditions, NO3- efflux was stimulated and was a function of root NO3- concentration. Conversely, at basic pH, NO3- uptake by NO3- and NO2(-)-induced and uninduced roots decreased, apparently because of the inhibition of influx. Calcium had little effect on NO3- uptake (influx) by NO2(-)-induced roots at either pH 3 or 6. However, in NO3(-)-induced roots, lack of Ca2+ at pH 3 significantly decreased net NO3- uptake and stimulated efflux. The results indicate that at acidic pH the decrease in net NO3- uptake is due to the stimulation of efflux, whereas at basic pH, it is due to the inhibition of influx.     

Diurnal variation in uptake and xylem contents of inorganic and assimilated N under continuous and interrupted N supply to Phleum pratense and Festuca pratensis

Compensation by dark-period uptake of NH(4)(+) and NO(3)(-) in the grasses Phleum pratense L. and Festuca pratensis Huds. following N deprivation during the preceding light period was investigated in flowing solution culture under an artificial 10/14 h light/dark cycle. N was supplied as either NO(3)(-), NH(4)(+) or NH(4)NO(3) at 20+/-5 mmol m(-3), available continuously or only during the dark period, for 5-10 d. Intermittent N supply did not affect total daily N uptake, growth rate or net partitioning of dry matter. Net uptake and influx of NO(3)(-) varied similarly throughout the diurnal cycle when NO(3)(-) was supplied continuously, with a marginal contribution by NO(3)(-) efflux. Influx was significantly higher and efflux slightly higher following interruption of NO(3)(-) supply during the light period. Nitrate accounted for 80% of N in xylem exudate except between hours 6-9 of the light period when the amino acid concentration increased 3-fold, primarily as glutamine. Diurnal variation in relative NO(3)(-) uptake exhibited five phases of constant acceleration/deceleration, described reasonably well assuming NO(3)(-) influx was subject to metabolic co-regulation by NO(3)(-) and amino acid levels in the cytoplasmic compartment of the roots. Accordingly, influx is determined by variation in root NO(3)(-) levels throughout the dark period and the first half of the light period, but is down-regulated by increased amino acid levels during the second half of the light period. The sharp light/dark transitions affect transpiration rate and hence xylem N flux which, in turn, affect NO(3)(-) levels in the cytoplasmic compartment of the roots and the rate of NO(3)(-) assimilation in the shoot.     

Ammonium and nitrate uptake by soybean during recovery from nitrogen deprivation

Soybean [Glycine max (L.) Merrill] plants that had been subjected to 15 d of nitrogen deprivation were resupplied for 10 d with 1.0 mol m-3 nitrogen provided as NO3-, NH4+, or NH4(+) + NO3- in flowing hydroponic culture. Plants in a fourth hydroponic system received 1.0 mol m-3 NO3- during both stress and resupply periods. Concentrations of soluble carbohydrates and organic acids in roots increased 210 and 370%, respectively, during stress. For the first day of resupply, however, specific uptake rates of nitrogen, determined by ion chromatography as depletion from solution, were lower for stressed than for non-stressed plants by 43% for NO3- resupply, by 32% for NH4(+) + NO3- resupply, and 86% for NH4+ resupply. When specific uptake of nitrogen for stressed plants recovered to rates for non-stressed plants at 6 to 8 d after nitrogen resupply, carbohydrates and organic acids in their roots had declined to concentrations lower than those of non-stressed plants. Recovery of nitrogen uptake capacity of roots thus does not appear to be regulated simply by the content of soluble carbon compounds within roots. Solution concentrations of NH4+ and NO3- were monitored at 62.5 min intervals during the first 3 d of resupply. Intermittent 'hourly' intervals of net influx and net efflux occurred. Rates of uptake during influx intervals were greater for the NH4(+)-resupplied than for the NO3(-)-resupplied plants. For NH4(+)-resupplied plants, however, the hourly intervals of efflux were more numerous than for NO3(-)-resupplied plants. It thus is possible that, instead of repressing NH4+ influx, increased accumulation of amino acids and NH4+ in NH4(+)-resupplied plants inhibited net uptake by stimulation of efflux on NH4+ absorbed in excess of availability of carbon skeletons for assimilation. Entry of NH4+ into root cytoplasm appeared to be less restricted than translocation of amino acids from the cytoplasm into the xylem.     

Deposition of ammonium and nitrate in the roots of maize seedlings supplied with different nitrogen salts

This study measured total osmolarity and concentrations of NH(4)(+), NO(3)(-), K(+), soluble carbohydrates, and organic acids in maize seminal roots as a function of distance from the apex, and NH(4)(+) and NO(3)(-) in xylem sap for plants receiving NH(4)(+) or NO(3)(-) as a sole N-source, NH(4)(+) plus NO(3)(-), or no nitrogen at all. The disparity between net deposition rates and net exogenous influx of NH(4)(+) indicated that growing cells imported NH(4)(+) from more mature tissue, whereas more mature root tissues assimilated or translocated a portion of the NH(4)(+) absorbed. Net root NO(3)(-) influx under Ca(NO(3))(2) nutrition was adequate to account for pools found in the growth zone and provided twice as much as was deposited locally throughout the non-growing tissue. In contrast, net root NO(3)(-) influx under NH(4)NO(3) was less than the local deposition rate in the growth zone, indicating that additional NO(3)(-) was imported or metabolically produced. The profile of NO(3)(-) deposition rate in the growth zone, however, was similar for the plants receiving Ca(NO(3))(2) or NH(4)NO(3). These results suggest that NO(3)(-) may serve a major role as an osmoticant for supporting root elongation in the basal part of the growth zone and maintaining root function in the young mature tissues.     

Shoot excision has no effect on net flux of protons, ammonium or nitrate in seedling roots of a conifer and three crop species

A high-flux region, 5mm from the root tips of seedlings of coastal Douglas-fir (Pseudotsuga menziesii), soybean (Glycine max), zucchini (Cucurbita pepo) and pea (Pisum sativum), was monitored using a microelectrode ion flux measurement system, for changes in the net fluxes of H(+), NH(4)(+) and NO(3)(-) in response to shoot removal. In all species, careful excision of the seedling shoot had no significant effect on the net fluxes of H(+), NH(4)(+) or NO(3)(-) measured 5mm from the root tip. Experiments were carried out for up to 80min after shoot removal, and no temporal interactions were noted.     

Ammonium and nitrate uptake by the floating plant Landoltia punctata

BACKGROUND AND AIMS: Plants from the family Lemnaceae are widely used in ecological engineering projects to purify wastewater and eutrophic water bodies. However, the biology of nutrient uptake mechanisms in plants of this family is still poorly understood. There is controversy over whether Lemnaceae roots are involved in nutrient uptake. No information is available on nitrogen (N) preferences and capacity of Landoltia punctata (dotted duckweed), one of the best prospective species in Lemnaceae for phytomelioration and biomass production. The aim of this study was to assess L. punctata plants for their ability to take up NH4+ and NO3- by both roots and fronds. METHODS: NO3- and NH4+ fluxes were estimated by a non-invasive ion-selective microelectrode technique. This technique allows direct measurements of ion fluxes across the root or frond surface of an intact plant. KEY RESULTS: Landoltia punctata plants took up NH4+ and NO3- by both fronds and roots. Spatial distribution of NH4+ and NO3- fluxes demonstrated that, although ion fluxes at the most distal parts of the root were uneven, the mature part of the root was involved in N uptake. Despite the absolute flux values for NH4+ and NO3- being lower in roots than at the frond surface, the overall capacity of roots to take up ions was similar to that of fronds because the surface area of roots was larger. L. punctata plants preferred to take up NH4+ over NO3- when both N sources were available. CONCLUSIONS: Landoltia punctata plants take up nitrogen by both roots and fronds. When both sources of N are available, plants prefer to take up NH4+, but will take up NO3- when it is the only N source.     

A comparison of NH4+ and NO3– net fluxes along roots of rice and maize

Net fluxes of NH₄⁺ and NO₃^– along adventitious roots of rice ( Oryza sativa L.) and the primary seminal root of maize ( Zea mays L.) were investigated under nonperturbing conditions using ion-selective microelectrodes. The roots of rice contained a layer of sclerenchymatous fibres on the external side of the cortex, whereas this structure was absent in maize. Net uptake of NH₄⁺ was faster than that of NO₃^– at 1 mm behind the apex of both rice and maize roots when these ions were supplied together, each at 0·1 mol m^–3. In rice, NH₄⁺ net uptake declined in the more basal regions, whereas NO₃^– net uptake increased to a maximum at 21 mm behind the apex and then it also declined. Similar patterns of net uptake were observed when NH₄⁺ or NO₃^– was the sole nitrogen source, although the rates of NO₃^– net uptake were faster in the absence of NH₄⁺. In contrast to rice, rates of NH₄⁺ and NO₃^– net uptake in the more basal regions of maize roots were similar to those near the root apex. Hence, the layer of sclerenchymatous fibres may have limited ion absorption in the older regions of rice roots.     

pH regulation in anoxic rice coleoptiles at pH 3.5: biochemical pHstats and net H+ influx in the absence and presence of NOFormula

During anoxia, cytoplasmic pH regulation is crucial. Mechanisms of pH regulation were studied in the coleoptile of rice exposed to anoxia and pH 3.5, resulting in H(+) influx. Germinating rice seedlings survived a combination of anoxia and exposure to pH 3.5 for at least 4 d, although development was retarded and net K(+) efflux was continuous. Further experiments used excised coleoptile tips (7-10 mm) in anoxia at pH 6.5 or 3.5, either without or with 0.2 mM NO(3)(-), which distinguished two processes involved in pH regulation. Net H(+) influx (μmol g(-1) fresh weight h(-1)) for coleoptiles with NO(3)(-) was ～1.55 over the first 24 h, being about twice that in the absence of NO(3)(-), but then decreased to 0.5-0.9 as net NO(3)(-) uptake declined from ～1.3 to 0.5, indicating reduced uptake via H(+)-NO(3)(-) symports. NO(3)(-) reduction presumably functioned as a biochemical pHstat. A second biochemical pHstat consisted of malate and succinate, and their concentrations decreased substantially with time after exposure to pH 3.5. In anoxic coleoptiles, K(+) balancing the organic anions was effluxed to the medium as organic anions declined, and this efflux rate was independent of NO(3)(-) supply. Thus, biochemical pHstats and reduced net H(+) influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5.     

Nitrate supply affects ammonium transport in canola roots

Plants may suffer from ammonium (NH4+) toxicity when NH4+ is the sole nitrogen source. Nitrate (NO3-) is known to alleviate NH4+ toxicity, but the mechanisms are unknown. This study has evaluated possible mechanisms of NO3- alleviation of NH4+ toxicity in canola (Brassica napus L.). Dynamics of net fluxes of NH4+, H+, K+ and Ca2+ were assessed, using a non-invasive microelectrode (MIFE) technique, in plants having different NO3- supplies, after single or several subsequent increases in external NH4Cl concentration. After an increase in external NH4Cl without NO3-, NH4+ net fluxes demonstrated three distinct stages: release (tau1), return to uptake (tau2), and a decrease in uptake rate (tau3). The presence of NO3- in the bathing medium prevented the tau1 release and also resulted in slower activation of the tau3 stage. Net fluxes of Ca2+ were in the opposite direction to NH4+ net fluxes, regardless of NO3- supply. In contrast, H+ and K+ net fluxes and change in external pH were not correlated with NH4+ net fluxes. It is concluded that (i) NO3- primarily affects the NH4+ low-affinity influx system; and (ii) NH4+ transport is inversely linked to Ca2+ net flux.     

Kinetics of NO3(-) net fluxes in Pinus pinaster, Rhizopogon roseolus and their ectomycorrhizal association, as affected by the presence of NO3(-) and NH4+

The impact of mineral N supply, N-free or NO3(-) with or without NH4+, on the subsequent uptake of NO3(-) by maritime pine seedlings associated with the ectomycorrhizal fungus Rhizopogon roseolus was studied using ion-selective microelectrodes. NO3(-) net fluxes into N-starved non-mycorrhizal short roots (NMSRs) were low and measurable only over the NO3(-) concentration range of 0-70 microM. The simple kinetics observed in those roots may reflect the dominant operation of a high-affinity NO3(-) transport system (HATS) which is constitutive. NO3(-) pretreatment increased the NO3(-) net fluxes and led to a complex kinetics that may reflect the operation of other HATS. A simple kinetics was observed in plants pre-incubated at high NH4+ concentration. In contrast, NO3(-) uptake kinetics presented only one saturation phase in the fungus, whether associated with the plant or not. NO3(-) uptake was greater after a pretreatment in N-free or NO3 (-) solution, but NH4+ pretreatment led to a threefold reduction in NO3 (-) uptake. These results suggest that the regulation of NO3(-) transport systems varies between the host and the fungal partner. This variation is likely to contribute to the positive effect of mycorrhizal association on N uptake in plants when the N supply is low and fluctuating.     

The effect of nitrogen nutrition on cluster root formation and proton extrusion by Lupinus albus

Nitrogen nutrition can influence cluster root formation in many wild species, but the effect of N form on cluster root formation and root exudation by white lupin is not known. In a solution culture study, we examined the effect of N nutrition (ammonium, nitrate, both or N2 fixation) on cluster root formation and H+ extrusion by white lupin plants under deficient and adequate P supply. The number of cluster roots increased greatly when plants were supplied with I microM P compared with 50 microM P, the increase being 7.8-fold for plants treated with (NH4)2SO4, 3-fold for plants treated with KNO3 and NH4NO3, and 2-4-fold for N2-fixing plants. Under P deficiency. NH4+-N supply resulted in production of a greater number and biomass of cluster roots than other N sources. Dry weight of cluster roots was 30 % higher than that of non-cluster roots in P-deficient plants treated with (NH4)2SO4 and NH4NO3. In plants treated with sufficient P (50 microM), the weight of non-cluster roots was approx. 90 % greater than that of cluster roots. Both total (micromol per plant h(-1)) and specific (micromol g(-1) root d. wt h(-1)) H+ extrusions were greatest from roots of plants supplied with (NH4)2SO4, followed by those supplied with NH4NO3 and N2 fixation, whereas plants receiving KNO3 had negative net H+ extrusion between the third and fifth week of growth (indicating uptake of protons or release of OH- ions). The rate of proton extrusion by NH4+-N-fed plants was similar under P-deficient and P-sufficient conditions. In contrast, proton exudation by N2-fixing plants and KNO3-treated plants was ten-fold greater under P deficiency than under P sufficiency. In comparison with P deficiency, plants treated with 50 microM P had a significantly higher concentration of P in roots, shoots and youngest expanded leaves (YEL). Compared with the N2 fixation and KNO3 treatments, total N concentration was highest in roots, shoots and YEL of plants supplied with (NH4)2SO4 and NH4NO3, regardless of P supply. Under P deficiency, K concentrations in roots decreased at all N supplies, especially in plants treated with (NH4)2SO4 and NH4NO3, which coincided with the greatest H+ extrusion at these P and N supplies. In conclusion, NH4-N nutrition stimulated cluster root formation and H+ extrusion by roots of P-deficient white lupin.     

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-.     

Effects of aluminium and mineral nutrition on growth and chemical composition of hydroponically grown seedlings of five different forest tree species

Forest die-back and impaired tree vitality have frequently been ascribed to Al-toxicity and Al-induced nutritional disorders due to increased acidification of forest soils. Therefore, in this experiment effects of Al were studied on growth and nutrient uptake with seedlings of five different forest tree species. During growth in culture solutions with and without Al all five species proved to be very Al-tolerant, despite high accumulation of Al in roots. In the coniferous evergreens Douglas-fir and Scots pine shoot as well as root Al concentrations were significantly higher than in the deciduous broad-leaved species oak and birch. Larch showed intermediate Al levels. In none of the five species did Al reduce nutrient concentrations or the Ca/Al ratio to values below the critical level. Besides differences in Al accumulation, coniferous and broad-leaved species also differed with respect to uptake and assimilation of nitrogen. Due to extra NH ₄ ⁺ uptake, oak and birch showed a much higher N uptake and higher NH ₄ ⁺ preference than the coniferous species. Especially with oak this high NH ₄ ⁺ preference in combination with a low specific root surface area resulted in a high root proton efflux density. In comparison to both broad-leaved trees and Scots pine the NO ₃ ⁻ reduction capacity of larch and Douglas-fir was extremely low. This may have important consequences for both species if grown in NO ₃ ⁻ -rich soils.     

Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea (Camellia sinensis) plants

BACKGROUND AND AIMS: Tea (Camellia sinensis) is considered to be acid tolerant and prefers ammonium nutrition, but the interaction between root zone acidity and N form is not properly understood. The present study was performed to characterize their interaction with respect to growth and mineral nutrition. METHODS: Tea plants were hydroponically cultured with NH4+, NO3- and NH(4+) + NO3-, at pH 4.0, 5.0 and 6.0, which were maintained by pH stat systems. KEY RESULTS: Plants supplied with NO3- showed yellowish leaves resembling nitrogen deficiency and grew much slower than those receiving NH4+ or NH(4+) + NO3- irrespective of root-zone pH. Absorption of NH4+ was 2- to 3.4-fold faster than NO3- when supplied separately, and 6- to 16-fold faster when supplied simultaneously. Nitrate-grown plants had significantly reduced glutamine synthetase activity, and lower concentrations of total N, free amino acids and glucose in the roots, but higher concentrations of cations and carboxylates (mainly oxalate) than those grown with NH4+ or NH(4+) + NO3-. Biomass production was largest at pH 5.0 regardless of N form, and was drastically reduced by a combination of high root-zone pH and NO3-. Low root-zone pH reduced root growth only in NO(3-)-fed plants. Absorption of N followed a similar pattern as root-zone pH changed, showing highest uptake rates at pH 5.0. The concentrations of total N, free amino acids, sugars and the activity of GS were generally not influenced by pH, whereas the concentrations of cations and carboxylates were generally increased with increasing root-zone pH. CONCLUSIONS: Tea plants are well-adapted to NH(4+)-rich environments by exhibiting a high capacity for NH4+ assimilation in their roots, reflected in strongly increased key enzyme activities and improved carbohydrate status. The poor plant growth with NO3- was largely associated with inefficient absorption of this N source. Decreased growth caused by inappropriate external pH corresponded well with the declining absorption of nitrogen.     

pH affects ammonium,nitrate and proton fluxes in the apical region of conifer and soybean roots

The effect of pH on nitrate and ammonium uptake in the high‐affinity transport system and low‐affinity transport system ranges was compared in two conifers and one crop species. Many conifers grow on acidic soils, thus their preference for ammonium vs nitrate uptake can differ from that of crop plants, and the effect of pH on nitrogen (N) uptake may differ. Proton, ammonium and nitrate net fluxes were measured at seedling root tips and 5, 10, 20 and 30 mm from the tips using a non‐invasive microelectrode ion flux measurement system in solutions of 50 or 1500 µM NH₄NO₃ at pH 4 and 7. In Glycine max and Pinus contorta, efflux of protons was observed at pH 7 while pH 4 resulted in net proton uptake in some root regions. Pseudotsuga menziesii roots consistently showed proton efflux behind the root tip, and thus appear better adapted to maintain proton efflux in acid soils. P. menziesii's ability to maintain ammonium uptake at low pH may relate to its ability to maintain proton efflux. In all three species, net nitrate uptake was greatest at neutral pH. Net ammonium uptake in G. max and net nitrate uptake in P. menziesii were greatly reduced at pH 4, particularly at high N concentration, thus N concentration should be considered when determining optimum pH for N uptake. In P. menziesii and G. max, net N uptake was greater in 1500 than 50 µM NH₄NO₃ solution, but flux profiles of all ions varied among species.     

Effects of a localized supply of nitrate on NO3 - uptake rate and growth of roots in Lolium multiflorum Lam.

Roots of higher plants are usually exposed to varying spatial and temporal changes in concentrations of soil mineral nitrogen. A split root system was used to see how Lolium multiflorum Lam. roots adapt to such variations to cope with their N requirements. Plants were grown in hydroponic culture with their root system split in two spatially separated compartments allowing them to be fed with or without KNO₃. Net NO₃ ^- uptake, ¹⁵NO₃ ^- influx and root growth were studied in relation to time. Within less than 24 h following deprivation of KNO₃ to half the roots, the influx in NO₃ ^- fed roots was observed to increase (about 200% of the influx measured in plant uniformly NO₃ ^- supplied control plant) thereby compensating the whole plant for the lack of uptake by the N deprived roots. Due to the large NO₃ ^- concentrations in the roots, the NO₃ ^- efflux was also increased so that the net uptake rate increased only slightly (35% maximum) compared with the values obtained for control plants uniformly supplied with NO₃ ^-. This increase in net NO₃ ^- uptake rate was not sufficient to compensate the deficit in N uptake rate of the NO₃ ^- deprived split root in the short term. Over a longer period (>1 wk), root growth of the part of the root system locally supplied with NO₃ ^- was stimulated. An increase in root growth was mainly responsable for the greater uptake of nitrate in Lolium multiflorum so that it was able to fully compensate the deficit in N uptake rate of the NO₃ ^- deprived split root.