High NH4+ efflux from roots of the common alpine grass,Festuca nigrescens,at field-relevant concentrations restricts net uptake

Alpine meadows of high ecological value could be severely endangered by anthropogenic N enrichment, modifying the relationships between species and the environment. While a constraint exerted by N availability on alpine plant development has been demonstrated by some fertilization experiments, in others no effect was observed. Basically, the problem is that mineral N absorption has not been characterized in alpine plants. In growth chamber experiments, we investigated the component fluxes of ¹⁵NO₃^? and ¹⁵NH₄⁺ uptake in a tussock grass (Festuca nigrescens) very common and representative of the dominant plant growth form in European alpine meadows. Rates of influx supported data already published for low elevation herbaceous species. These rates were up to ten times higher for NH₄⁺ than for NO₃^? but rates of net uptake were similar for both ions demonstrating the occurrence of elevated NH₄⁺ efflux (80% of primary influx). An increase in external N in the range of field-relevant concentrations did not substantially enhance net uptake. Thus, the alpine plant which is assumed to be adapted to relatively high soil NH₄⁺ responded like an NH₄⁺-sensitive species: as if it was unable to use the incoming nitrogen. It is suggested that the ability of this typical alpine grass to respond to increasing N availability due to global changes is limited.     

The influence of monovalent cations upon influx and efflux of Ca2+ in barley roots

The effects of monovalent cations, inhibitors of metabolism dinitrophenol (DNP), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and KCN and temperature variations upon Ca²⁺ fluxes in intact roots of barley (Hordeum vulgare L. cv. Fergus and Herta) seedlings were investigated. ⁴⁵Ca²⁺ influx was depressed in CaSO₄-grown (low-salt) plants by the presence of NH₄⁺, K⁺, or Na⁺ in the uptake medium. In contrast Ca²⁺ influx was slightly increased by Li⁺. In low-salt roots pretreated with KCN and in roots preloaded with K⁺ (high-K⁺ plants), the presence of K⁺ in the medium had no significant effect on Ca²⁺ influx, while in roots preloaded with Na⁺, the presence of K⁺ in the medium depressed Ca²⁺ influx. In absolute terms, Ca²⁺ influx was significantly greater in high-salt (both K⁺ or Na⁺ preloaded) than in low-salt roots.Patterns of ⁴⁵Ca²⁺ efflux in the absence and in the presence of K⁺, NH₄⁺, or Li⁺ in the external medium showed that these monovalent cations caused stimulation of ⁴⁵Ca²⁺ efflux both from the cytoplasmic and vacuolar phases.It was noted that these modifications of Ca²⁺ fluxes by monovalent cations are transient and characteristic of a transitional stage of cation uptake by low-salt roots. We conclude that, together with stimulated active H⁺ efflux (another characteristic of this transitional stage), modifications of Ca²⁺ fluxes during monovalent cation uptake by low-salt roots is a response directed towards the maintenance of electrical neutrality.Determination of net fluxes revealed that the plants were close to Ca²⁺ flux equilibrium in the growth medium (0.5 mM CaSO₄). Transfer of these plants to 0.5 mM CaSO₄ + 0.25 mM K₂SO₄ caused a net release of CA²⁺ into the external medium.     

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.     

Complexity of potassium acquisition: How much flows through channels?

The involvement of potassium (K⁺)-selective, Shaker-type channels, particularly AKT1, in primary K⁺ acquisition in roots of higher plants has long been of interest, particularly in the context of low-affinity K⁺ uptake, at high K⁺ concentrations, as well as uptake from low-K⁺ media under ammonium (NH₄⁺) stress. We recently demonstrated that K⁺ channels cannot mediate K⁺ acquisition in roots of intact barley (Hordeum vulgare L.) seedlings at low (22.5 µM) external K⁺ concentrations ([K⁺]_ext) and in the presence of high (10 mM) external NH₄⁺, while the model species Arabidopsis thaliana L. utilizes channels under comparable conditions. However, when external NH₄⁺ was suddenly withdrawn, a thermodynamic shift to passive (channel-mediated) K⁺ influx was observed in barley and both species demonstrated immediate and dramatic stimulations in K⁺ influx, illustrating a hitherto unexplored magnitude and rapidity of K⁺-uptake capacity and plasticity. Here, we expand on our previous work by offering further characterization of channel-mediated K⁺ fluxes in intact barley, with particular focus on anion effects, root respiration and pharmacological sensitivity and highlight key additions to the current model of K⁺ acquisition.     

Optimization of ammonium acquisition and metabolism by potassium in rice (Oryza sativa L. cv. IR-72)

We present the first characterization of K⁺ optimization of N uptake and metabolism in an NH₄⁺‐tolerant species, tropical lowland rice (cv. IR‐72). ¹³N radiotracing showed that increased K⁺ supply reduces futile NH₄⁺ cycling at the plasma membrane, diminishing the excessive rates of both unidirectional influx and efflux. Pharmacological testing showed that low‐affinity NH₄⁺ influx may be mediated by both K⁺ and non‐selective cation channels. Suppression of NH₄⁺ influx by K⁺ occurred within minutes of increasing K⁺ supply. Increased K⁺ reduced free [NH₄⁺] in roots and shoots by 50–75%. Plant biomass was maximized on 10 mm NH₄⁺ and 5 mm K⁺, with growth 160% higher than 10 mm NO₃^‐‐grown plants, and 220% higher than plants grown at 10 mm NH₄⁺ and 0.1 mm K⁺. Unlike in NH₄⁺‐sensitive barley, growth optimization was not attributed to a reduced energy cost of futile NH₄⁺ cycling at the plasma membrane. Activities of the key enzymes glutamine synthetase and phosphoenolpyruvate carboxylase (PEPC) were strongly stimulated by elevated K⁺, mirroring plant growth and protein content. Improved plant performance through optimization of K⁺ and NH₄⁺ is likely to be of substantial agronomic significance in the world's foremost crop species.     

Caesium,potassium and ammonium distributions in different organs of tropical plants

In the present work the distribution of ions in aboveground plant parts was studied in order to establish the suitability of using radiocaesium as a tracer for the plant absorption of nutrients, such as potassium (K⁺) and ammonium (NH₄⁺). We present the results for the distributions of ¹³⁷Cs, ⁴⁰K and NH₄⁺ from four tropical plant species: lemon (Citrus aurantifolia), orange (Citrus sinensis), guava (Psidium guajava) and chili pepper (Capsicum frutescens). Activity concentrations of ¹³⁷Cs and ⁴⁰K were measured by gamma spectrometry and concentrations of free NH₄⁺ ions by a colorimetric method. Similarly to potassium and ammonium, caesium showed a high mobility within the plants, exhibiting the highest values of concentration in the growing parts of the tree (fruits, new leaves, twigs, and barks). A significant correlation between activity concentrations of ¹³⁷Cs and ⁴⁰K was observed in these tropical plants. The K/Cs discrimination ratios were approximately equal to unity in different compartments of each individual plant, suggesting that caesium could be a good tracer for ⁴⁰K in tropical woody fruit species. Despite the similarity observed for the behaviour of caesium and ammonium in the newly grown plant compartments, ¹³⁷Cs was not well correlated with NH₄⁺. Significant temporal changes in the NH₄⁺ concentrations were observed during the development of fruits, while the ¹³⁷Cs activity concentration alterations were not of great importance, indicating, therefore, that Cs⁺ and free NH₄⁺ ions could have distinct concentration ratios for each particular plant organ.     

Effects of NH4+ concentration on growth,morphology and NH4+ uptake kinetics of Salvinia natans

Many plants develop toxicity symptoms and have reduced growth rates when supplied with ammonium (NH₄⁺) as the only source of inorganic nitrogen. In the present study, the growth, morphology, NH₄⁺ uptake kinetics and mineral concentrations in the tissues of the free-floating aquatic plant Salvinia natans (water fern) supplied exclusively with NH₄⁺–N at concentrations of 0.25–15 mM were investigated. S. natans grew well, with relative growth rates of c. 0.25 g g^?1 d^?1 at external NH₄⁺ concentrations up to 5 mM, but at higher levels growth was suppressed and the plants had small leaves and short roots with stunted growth. The high-affinity transport system (HATS) that mediate NH₄⁺ uptake at dilute NH₄⁺ levels was downregulated at high NH₄⁺ concentrations with lower velocities of maximum uptake (V_max) and higher half-saturation constants (K_1/2). High NH₄⁺ levels also barely affected the concentrations of mineral cations and anions in the plant tissue. It is concluded that S. natans can be characterized as NH₄⁺-tolerant in line with a number of other species of wetland plants as growth was unaffected at NH₄⁺ concentrations as high as 5 mM and as symptoms of toxicity at higher concentrations were relatively mild. Depolarization of the plasma membrane to the equilibrium potential for NH₄⁺ at high external concentrations may be a mechanism used by the plant to avoid excessive futile transmembrane cycling. S. natans is tolerant to the high NH₄⁺ levels that prevail in domestic and agricultural wastewaters, and the inherent high growth rate and the ease of biomass harvesting make S. natans a primary candidate for use in constructed wetland systems for the treatment of various types of nitrogen-rich wastewaters.     

High irradiance increases NH(4)(+) tolerance in Pisum sativum: Higher carbon and energy availability improve ion balance but not N assimilation

The widespread use of NO₃⁻ fertilization has had a major ecological impact. NH₄⁺ nutrition may help to reduce this impact, although high NH₄⁺ concentrations are toxic for most plants. The underlying tolerance mechanisms are not yet fully understood, although they are thought to include the limitation of C, the disruption of ion homeostasis, and a wasteful NH₄⁺ influx/efflux cycle that carries an extra energetic cost for root cells.In this study, high irradiance (HI) was found to induce a notable tolerance to NH₄⁺ in the range 2.5-10 mM in pea plants by inducing higher C availability, as shown by carbohydrate content. This capacity was accompanied by a general lower relative N content, indicating that tolerance is not achieved through higher net N assimilation on C-skeletons, and it was also not attributable to increased GS content or activity in roots or leaves. Moreover, HI plants showed higher ATP content and respiration rates. This extra energy availability is related to the internal NH₄⁺ content regulation (probably NH₄⁺ influx/efflux) and to an improvement of the cell ionic balance.The limited C availability at lower irradiance (LI) and high NH₄⁺ resulted in a series of metabolic imbalances, as reflected in a much higher organic acid content, thereby suggesting that the origin of the toxicity in plants cultured at high NH₄⁺ and LI is related to their inability to avoid large-scale accumulation of the NH₄⁺ ion.     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

The effect of environmental ammonia concentrations on the ion-exchange of shore crabs,Carcinus maenas (L.)

1. Ion exchange characteristics (ion-permeability, net, passive and active ion fluxes) of adult shore crabs were studied in relation to experimentally-increased external ammonia concentrations.2. Moderately-elevated ammonia concentrations (up to about 1 mmol/1 NH₄⁺) induce an increase in the ion permeability and the salt fluxes across the body wall of Carcinus maenas. At still higher NH₄⁺ concentrations, ion-permeability and ion fluxes are reduced again.3. Active salt influx generally follows the same pattern as observed for whole animal permeability, probably because both parameters are strongly related to gill perfusion and gill ventilation.4. Prolonged exposure to elevated ammonia concentrations in the environment is unfavourable, not because of the NH₄⁺ toxicity, but because of the higher energy requirements associated with the higher salt fluxes.     

Interactions between K+ and NH4+: effects on ion uptake by rice roots

Interactive effects of K⁺ and N (principally NH₄⁺) on plant growth and ion uptake were investigated using hydroponically grown rice (Oryza sativa L. cv. M202) seedlings by varying the availability of NH₄⁺ or NO₃^? and K⁺ during an 18d growth period, a 3d pretreatment period and during flux measurements. Plants grew best in media containing 100 mmol m^?3 NH₄⁺ and 200mmolm^?3 K⁺ (N100/K200), followed by N2/K200 < N100/K2 < N2/K2. ⁸⁶Rb⁺(K⁺) fluxes were increased by exposure to N during the 18 d growth period and the 3 d of pretreatment, but decreased by the presence of NH₄⁺ during flux measurements. This inhibition was a function of prior N/K provision and the [NH₄⁺]₀ present during flux determinations. NH₄⁺ was least inhibitory to 86Rb⁺(K⁺) influx in high-N/low-K plants. Pretreatments with K⁺ failed to stimulate NH₄⁺ uptake, and the presence of K⁺ in the uptake solutions reduced NH₄⁺ fluxes only in high-N/low-K plants.     

Influence of Nitrate and Ammonium Nutrition on the Uptake, Assimilation, and Distribution of Nutrients in Ricinus communis

Ricinus communis L. plants were grown in nutrient solutions in which N was supplied as NO₃⁻ or NH₄⁺, the solutions being maintained at pH 5.5. In NO₃⁻-fed plants excess nutrient anion over cation uptake was equivalent to net OH⁻ efflux, and the total charge from NO₃⁻ and SO₄²⁻ reduction equated to the sum of organic anion accumulation plus net OH⁻ efflux. In NH₄⁺-fed plants a large H⁺ efflux was recorded in close agreement with excess cation over anion uptake. This H⁺ efflux equated to the sum of net cation (NH₄⁺ minus SO₄²⁻) assimilation plus organic anion accumulation. In vivo nitrate reductase assays revealed that the roots may have the capacity to reduce just under half of the total NO₃⁻ that is taken up and reduced in NO₃⁻-fed plants. Organic anion concentration in these plants was much higher in the shoots than in the roots. In NH₄⁺-fed plants absorbed NH₄⁺ was almost exclusively assimilated in the roots. These plants were considerably lower in organic anions than NO₃⁻-fed plants, but had equal concentrations in shoots and roots. Xylem and phloem saps were collected from plants exposed to both N sources and analyzed for all major contributing ionic and nitrogenous compounds. The results obtained were used to assist in interpreting the ion uptake, assimilation, and accumulation data in terms of shoot/root pH regulation and cycling of nutrients.     

Nitrogen Acquisition from Atmospheric NH3 by Lolium perenne

Ammonia (NH₃) is the third most abundant N species in the atmosphere and, due to various natural and anthropogenic sources, can reach high concentrations in some areas. While some plants show effects of toxicity, others are capable of using this N-form and grow well without any utilization of soil-N. Acquisition of atmospheric NH₃ will affect the acid-base balance of the plants as absorption and dissolution causes an alkalinisation (production of OH^?) and assimilation of NH₃ results in an acidification (generation of H⁺). As there is only a limited capacity for biochemical disposal of excess H⁺ in shoots, pH regulation may involve H⁺/OH^? extrusion into the media via roots and transport of (in)organic ions between roots and above-ground parts of the plant. Our aim therefore was to assess NH₃ acquisition by Lolium perenne and to study the effects of gas phase NH₃ on growth, acid-base balance and mineral composition of the plants. The experiments therefore included application of a range of ¹⁴NH₃ to the shoots and of ¹⁵N as NO₃^?, NH₄⁺ or NH₄NO₃ to the roots, from which the amount of gas phase NH₃ acquisition could be quantified. Analysis of the mineral composition provided data for calculation of acid-base balance as well as for water use efficiencies of the plants. The results indicate that over the range of NH₃ supplied, plants from all treatments could utilize gas-phase NH₃ as demonstrated by increases in growth and in N and C use efficiencies. Plants receiving NO₃^? via their roots had a higher capacity to use gaseous NH₃ than those growing with NH₄⁺. NH₃ assimilation in shoots reduced both the acid load with NH₄⁺ nutrition and the alkaline load with NO₃^? supply to the roots. The results of the experiments are discussed in relation to possible acid-base regulation mechanisms of the whole plant.     

Rapid Ammonia Gas Transport Accounts for Futile Transmembrane Cycling under NH3/NH4+ Toxicity in Plant Roots

Futile transmembrane NH₃/NH₄⁺ cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH₃/NH₄⁺ toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH₃ or NH₄⁺) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope ¹³N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH₃ species, rather than the NH₄⁺ ion. Influx of ¹³NH₃/¹³NH₄⁺, which exceeded 200 µmol g^–1 h^–1, was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH₃ transport. Influx followed Michaelis-Menten kinetics for NH₃ (but not NH₄⁺), as a function of external concentration (K_m = 152 µm, V_max = 205 µmol g^–1 h^–1). Efflux of ¹³NH₃/¹³NH₄⁺ responded with a nearly identical K_m. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH₃ is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions.Ammonia/ammonium (NH₃/NH₄⁺) toxicity in higher plants has resulted in crop reduction and forest decline (Pearson and Stewart, 1993; Vitousek et al., 1997; Britto and Kronzucker, 2002), biodiversity loss (Stevens et al., 2004; Bobbink et al., 2010), and species extirpation (de Graaf et al., 1998; McClean et al., 2011). These major ecological and economic problems have been aggravated by an accelerated global nitrogen (N) cycle caused primarily by the industrialized production and use of N fertilizers (Galloway et al., 2008; Gruber and Galloway, 2008). With increasing global population and demands on agricultural production, there is no sign of this trend easing: anthropogenic N fixation has reached 210 teragrams year^–1, an approximately 12% increase from 2005 and an approximately 1,300% rise from 150 years ago (Galloway et al., 2008; Fowler et al., 2013).Although considerable knowledge of the causes and mechanisms of NH₃/NH₄⁺ toxicity has accrued in recent years, our understanding of the key processes remains rudimentary (Gerendas et al., 1997; Britto and Kronzucker, 2002). A major hypothesis is that of futile transmembrane NH₄⁺ cycling, which proposes a pathological inability of root cells to restrict the primary entry of NH₄⁺ at high external concentrations ([NH₄+]_ext); many downstream toxicological events are contingent upon this entry (Britto et al., 2001b). In this model, a rapid, thermodynamically passive influx of NH₄⁺ is coupled to an active efflux of NH₄⁺ that is nearly as rapid, constraining normal cellular function and energetics and resulting in plant growth decline and mortality. This phenomenon is thought to occur in NH₄⁺-sensitive species such as barley (Hordeum vulgare) and, to a lesser extent, in tolerant species such as rice (Oryza sativa), which can be susceptible at higher thresholds (Balkos et al., 2010; Chen et al., 2013).Most soils are typically acidic, especially when [NH₄⁺] is high (i.e. in the millimolar range; Van Breemen et al., 1982; Bobbink et al., 1998; Britto and Kronzucker, 2002), and given the pKa of 9.25 for the conjugate pair NH₃/NH₄⁺, [NH₃] is generally low (Izaurralde et al., 1990; Weise et al., 2013). Consequently, the fluxes of NH₃ have largely been considered negligible (Britto et al., 2001a; Britto and Kronzucker, 2002; Loqué and von Wirén, 2004), in contrast to NH₄⁺ fluxes, which are well characterized physiologically (Lee and Ayling, 1993; Wang et al., 1993a, 1993b; Kronzucker et al., 1996) and at the molecular level (Rawat et al., 1999; von Wirén et al., 2000; Ludewig et al., 2007), at least at lower concentrations. However, the transport of NH₃ across membranes has received new attention in the light of evidence that some members of the aquaporin (AQP) family of transporters, a diverse and ubiquitous class of major intrinsic proteins (Maurel et al., 2008; Hove and Bhave, 2011), can mediate NH₃ fluxes in single-cell systems (Jahn et al., 2004; Holm et al., 2005; Loqué et al., 2005; Saparov et al., 2007). However, a convincing demonstration that AQPs transport NH₃ in planta is currently lacking. Given the unusually high capacity of AQP-mediated fluxes relative to those of ion channels and other transporters (Kozono et al., 2002), it is possible that sizable NH₃ fluxes can be conducted through AQPs, even at very low external NH₃ concentration ([NH₃]_ext).Here, we have critically reexamined the hypothesis that futile cycling is composed of cationic NH₄⁺ fluxes across the plasmalemma, of which an active efflux mechanism accounts for energetic demands directly contributing to toxicity (Britto et al., 2001b). We present evidence for the following alternative scenario: 1) futile cycling consists mainly of the passive electroneutral flux of the conjugate base NH₃; 2) such fluxes rapidly span both major membrane systems in root cells (i.e. plasmalemma and tonoplast); 3) AQPs mediate such fluxes; and 4) a thermodynamic equilibrium of NH₃ is established throughout the cell, resulting in hyperaccumulation of NH₄⁺ in the acidic vacuole. This evidence comes primarily from positron emission tracing with the short-lived radioisotope ¹³N, used to characterize the component fluxes of futile cycling at the cellular level in the model species barley. We have coupled this with ⁴²K⁺ radiotracing, to provide comparison with a well-understood cationic flux, as well as electrophysiological, respiratory, pharmacological, and histochemical analyses.     

Simultaneous measurement of ammonium,nitrate and proton fluxes along the length of eucalypt roots

Knowledge of the preferred source of N for Eucalyptus nitens will lead to improved fertiliser management practices in plantations. Ion selective microelectrodes were used non-invasively to measure simultaneously net fluxes of NH₄ ⁺, NO₃ ^– and H⁺ along the tap root of solution-cultured E. nitens. Measurements were conducted in solutions containing 100 m NH₄NO₃. The pattern of fluxes was such that there was a large influx of NH₄ ⁺, a smaller influx of NO₃ ^– and large H⁺ efflux. The ratio of these fluxes was constant, according to the ratio 3:1:–6 (NH₄ ⁺:NO₃ ^–:H⁺). Within the region 20–60 mm from the root apex of E. nitens seedlings there was spatial and temporal variation in fluxes but flux patterns remained constant. Root hair density did not affect fluxes nor did proximity to lateral roots. Variation was less than that found in previous studies of localised root fluxes using similar high-resolution measurement techniques. It was concluded that small-scale spatial variation in fluxes may have confounded previous studies. There were associations between fluxes of all three ions, the strongest associations being between NH₄ ⁺ and H⁺, and NH₄ ⁺ and NO₃ ^–. Overall, these results are consistent with NH₄ ⁺ being the preferred source N for E. nitens.     

Net fluxes of ammonium and nitrate in association with H+ fluxes in fine roots of Populus popularis

Poplar plants are cultivated as woody crops, which are often fertilized by addition of ammonium (NH₄ ⁺) and/or nitrate (NO₃ ^?) to improve yields. However, little is known about net NH₄ ⁺/NO₃ ^? fluxes and their relation with H⁺ fluxes in poplar roots. In this study, net NH₄ ⁺/NO₃ ^? fluxes in association with H⁺ fluxes were measured non-invasively using scanning ion-selective electrode technique in fine roots of Populus popularis. Spatial variability of NH₄ ⁺ and NO₃ ^? fluxes was found along root tips of P. popularis. The maximal net uptake of NH₄ ⁺ and NO₃ ^? occurred, respectively, at 10 and 15 mm from poplar root tips. Net NH₄ ⁺ uptake was induced by ca. 48 % with provision of NO₃ ^? together, but net NO₃ ^? uptake was inhibited by ca. 39 % with the presence of NH₄ ⁺ in poplar roots. Furthermore, inactivation of plasma membrane (PM) H⁺-ATPases by orthovanadate markedly inhibited net NH₄ ⁺/NO₃ ^? uptake and even led to net NH₄ ⁺ release with NO₃ ^? co-provision. Linear correlations were observed between net NH₄ ⁺/NO₃ ^? and H⁺ fluxes in poplar roots except that no correlation was found between net NH₄ ⁺ and H⁺ fluxes in roots exposed to NH₄Cl and 0 mM vanadate. These results indicate that root tips play a key role in NH₄ ⁺/NO₃ ^? uptake and that net NH₄ ⁺/NO₃ ^? fluxes and the interaction of net fluxes of both ions are tightly associated with H⁺ fluxes in poplar roots.     

Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and is linked to NH4+ efflux and GMPase activity

Root growth in higher plants is sensitive to excess ammonium (NH₄⁺). Our study shows that contact of NH₄⁺ with the primary root tip is both necessary and sufficient to the development of arrested root growth under NH₄⁺ nutrition in Arabidopsis. We show that cell elongation and not cell division is the principal target in the NH₄⁺ inhibition of primary root growth. Mutant and expression analyses using DR5:GUS revealed that the growth inhibition is furthermore independent of auxin and ethylene signalling. NH₄⁺ fluxes along the primary root, measured using the Scanning Ion‐selective Electrode Technique, revealed a significant stimulation of NH₄⁺ efflux at the elongation zone following treatment with elevated NH₄⁺, coincident with the inhibition of root elongation. Stimulation of NH₄⁺ efflux and inhibition of cell expansion were significantly more pronounced in the NH₄⁺‐hypersensitive mutant vtc1‐1, deficient in the enzyme GDP‐mannose pyrophosphorylase (GMPase). We conclude that both restricted transmembrane NH₄⁺ fluxes and proper functioning of GMPase in roots are critical to minimizing the severity of the NH₄⁺ toxicity response in Arabidopsis.     

Acid-base regulation during ammonium assimilation in Hydrodictyon africanum

Abstract The acid-base balance during ammonium (used to mean NH ₄⁺ and/or NH₃) assimilation in Hydrodictyon africanum has been measured on cells growing with about 1 mol m^?3 ammonium at an external pH of about 6.5. Measurements made included (1) ash alkalinity (corrected for intracellular ammonium) which yields net organic negative charge, (2) the accumulation of organic N in the cells and (3) the change in extracellular H⁺ (from the pH change and the buffer capacity). These measurements showed that some 0.25 excess organic negative charge (half in the cell wall, half inside the plasmalemma) accumulates per organic N synthesized, while some 1.25H⁺ accumulate in the medium per organic N synthesized. Granted a permeability (PNH₃) of some 10^?3 cm s^?1, and a finite [NH₃] in the cytoplasm of these N-assimilating cells it is likely that most of the ammonium entering these growing cells is as NH ₄⁺. This means that most of the H ⁺ appearing in the medium must have originated from inside the cell and have been subjected to active efflux at the plasmalemma: H⁺ accumulates in the medium equivalent to any NH₃ entry by requilibration from exogenous NH ₄⁺. The cell composition (net organic negative charge, organic N content) is very similar in these ammonium-grown cells to that of NO₃⁺grown cells, suggesting that there is no action of a ‘biochemical pH stat’ during longterm assimilation of NO₃⁺in H. africanum. Short-term experiments were carried out at an external pH of 7.2 in which ammonium at various concentrations were supplied to NO₃⁺-grown cells. There was in all cases a rapid influx followed by a slower uptake; at least at the lower concentrations (less than 100 μmol dm^?3) the net influx was all attributable to NH₄⁺influx via a uniporter, probably partly short-circuited by a passive NH₃ efflux due to intrinsic membrane permeability to NH₃. The net ammonium influx was in all cases associated with H⁺ accumulation in the medium. (1.3-1.7 H ⁺ per ammonium taken up); as in the growth experiments, most of the ammonium taken up was assimilated. Determinations of cytoplasmic pH showed either no effect on, or a slight decrease in, pH during ammonium assimilation; the changes that occurred were in the direction expected for actuating a ‘pH-regulating’ change in H⁺ fluxes.