Depletion of the heaviest stable N isotope is associated with NH₄⁺/NH₃ toxicity in NH₄⁺-fed plants

Abstract

Background

In plants, nitrate (NO₃^-) nutrition gives rise to a natural N isotopic signature (δ¹⁵N), which correlates with the δ¹⁵N of the N source. However, little is known about the relationship between the δ¹⁵N of the N source and the ¹⁴N/¹⁵N fractionation in plants under ammonium (NH₄⁺) nutrition. When NH₄⁺ is the major N source, the two forms, NH₄⁺ and NH₃, are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH₃ (g) and NH₄⁺ (aq) which drives to a different δ¹⁵N. Nine plant species with different NH₄⁺-sensitivities were cultured hydroponically with NO₃^- or NH₄⁺ as the sole N sources, and plant growth and δ¹⁵N were determined. Short-term NH₄⁺/NH₃ uptake experiments at pH 6.0 and 9.0 (which favours NH₃ form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH₄⁺ and NH₃.

Results

Several NO₃^--fed plants were consistently enriched in ¹⁵N, whereas plants under NH₄⁺ nutrition were depleted of ¹⁵N. It was shown that more sensitive plants to NH₄⁺ toxicity were the most depleted in ¹⁵N. In parallel, N-deficient pea and spinach plants fed with ¹⁵NH₄⁺ showed an increased level of NH₃ uptake at alkaline pH that was related to the ¹⁵N depletion of the plant. Tolerant to NH₄⁺ pea plants or sensitive spinach plants showed similar trend on ¹⁵N depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO₃ as control discarded that the differences observed arise from pH detrimental effects.

Conclusions

This article proposes that the negative values of δ¹⁵N in NH₄⁺-fed plants are originated from NH₃ uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH₄⁺/NH₃ toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH₄⁺ may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionation.

Background

Nitrogen (N) and carbon (C) are the main components of all living organisms and regulate the productivity of most ecosystems. In agriculture, N is by far the main nutrient in fertilisers, with nitrate (NO₃^-) and ammonium (NH₄⁺) being the main N sources used by plants. However, relatively little is known about the isotopic fractionation during uptake of these ions. Assessment under natural conditions is difficult because, under most circumstances, NO₃^- and NH₄⁺ are simultaneously present in the soil and their concentrations change both spatially and temporally over a wide range (e.g., 20 μM to 20 mM) [1, 2]. Furthermore, this situation becomes even more complex if the rhizosphere and its symbiotic interactions (N₂-fixing organisms or mycorrhiza) are taken into account.

The natural variation in stable N isotopes has been shown to be a powerful tool in several studies of plant and ecosystem N dynamics [3]. Generally, the global δ¹⁵N value of the plant biomass is determined by that of the primary N source (soil N, fertiliser, N₂) [4]. Some studies assume that the δ¹⁵N of leaf tissue reflects that of the source in the soil (e.g., see [5]). This assumption implies that the isotope ratio of the N source is preserved during N absorption, assimilation and translocation. However, it is clear that physiological processes and biological mechanisms, such as N-uptake, assimilation through distinct pathways, internal N recycling in the plant and gaseous N exchange, can discriminate against ¹⁵N [4]. Furthermore, plant N fractionation is also dependent on the N availability. Thus, in the case of unlimited substrate (N) availability, an isotope effect will always be expressed, and therefore, the arising δ¹⁵N will be lower than in the N source if fractionation occurs [6]. In contrast, in a growth system where the quantity of substrate (N) is limited, and the organism exhausts the N source completely, the plant δ¹⁵N will be similar (or even identical) to the original N source [6, 7]. Most studies concerning physiological and natural N fractionation have involved plants grown with NO₃^- as the only N source. A review of these studies [6] showed that N fractionation changes with plant age, the external NO₃^- concentration and the partitioning of N metabolism between the roots and shoots.

Similarly to NO₃^-, NH₄⁺ influx through the membrane of plant cells exhibits a predominantly biphasic pattern. Thus, at concentrations up to 0.5-1 mM N, influx occurs via the high affinity transport system (HATS), which is saturable and energy dependent and has a K _m in the submillimolar concentration range; the non-saturable low affinity transport system (LATS) operates with a K _m in the millimolar concentration range, i.e., at N concentrations above 0.5-1 mM, for most plant roots [8, 9].

While the proteins responsible for the high-affinity NH₄⁺ transporters have been identified in many plant species, the low-affinity uptake system proteins have yet to be identified [9]. Recently, Loqué and von Wirén reviewed the different levels at which NH₄⁺ transport is regulated in plant roots under HATS conditions [10]. A functional analysis of several ammonium transporters (AMTs) expressed in Xenopus oocytes showed evidence that NH₄⁺, rather than NH₃, uniport is the most likely transport mechanism for AMT1-type transporters from plants [11–13]. Nevertheless, individual plant AMT/Rh transporters may use different transport mechanisms [13] compared with the AMT2-type transporters, which recruit NH₄⁺-mediated electroneutral NH₄⁺ transport, probably in the form of NH₃[14, 15].

On the contrary, the molecular basis of transport under LATS conditions remains poorly understood. LATS for NH₄⁺ operates when NH₄⁺ is present at high concentrations in solution; under these conditions, several symptoms of toxicity have often been observed in a broad range of plant species [2]. Few studies have examined the natural isotopic signature of plants grown with NH₄⁺ nutrition under LATS conditions and its relationship with sensitivity or tolerance to NH₄⁺ nutrition. It has been speculated that NH₃ could be the chemical species that enters the plant from the external medium via the plasma membrane [7, 16]. Under conditions of high external pH and high NH₄⁺, the transport of NH₃ across membranes occurs, and it can become biologically significant [16, 17]. In agro-ecosystems, in which the soils are currently fertilised with urea (50% of the total world fertiliser N consumption [18]) or (NH₄)₂SO₄, emissions of N in the NH₃ form take place (i.e., up to 10-20% of N in fertilisers applied as urea may be lost in the soil [19]). Thus, under these conditions, significant amounts of NH₃ may be present in the soil and therefore enter the plant. When NH₄⁺ is applied as the only N source or NH₄⁺ is formed naturally in soils via mineralization of organic matter, the two forms, NH₄⁺ and NH₃, are present in the nutrient solution. The neutral and ionic forms do not have exactly the same natural isotopic signatures because there is a 1.025 thermodynamic isotope effect between NH₃ (g) and NH₄⁺ (aq), so NH₃ (aq) is depleted for ¹⁵N by 20‰ relative to NH₄⁺ (aq) [20]; in addition, the equilibrium fractionation factor for exchange of NH₃ (aq) with NH₃ (g) has been estimated as ~ 1.005 [21].

Thus, an understanding of the physiological processes that lead to variations in the stable isotopic composition is required. This work was intended to assess the natural δ¹⁵N dynamics for several plant species grown hydroponically under controlled conditions and with only one N source, namely NO₃^- or NH₄⁺. Our working hypothesis for this study was that a part of NH₄⁺ enters the plant root as neutral molecules (i.e. NH₃) favouring the isotopic fractionation and this fractionation process during NH₄⁺ uptake is related to the sensitivity of plants to NH₄⁺ nutrition. Fractionation of the N source throughout the whole plant was interpreted on the basis of the relative transport of NH₄⁺ and NH₃. We also propose that LATS for NH₄⁺ uptake may have two components, one that involves the ionic form (NH₄⁺) and another that involves the molecular form (NH₃).

Methods

Plant Culture

i) Isotopic signature experiment in several plant species

Nine species that show different NH₄⁺ tolerances were grown hydroponically with NH₄⁺ or NO₃^- as the sole N sources. Lettuce (Lactuca sativa L. cv. Marine), spinach (Spinacia oleracea L. cv. Spinner), tomato (Solanum lycopersicum L. cv. Trust), pea (Pisum sativum L. cv. Eclipse) and lupin (Lupinus albus L. cv. albus) plants were germinated, cultured and treated as described previously [22]. Carob (Ceratonia siliqua sp.) and Acacia aneura sp. plants were grown according to [23]. Perennial ryegrass (Lolium perenne L. cv. Herbus) and white clover (Trifolium repens L. cv. Huia) were cultured according to [24]. Pea plants (cv. Sugar-snap) were grown according to [25], and spinach (cv. Gigante de invierno) and pea plants (cv. Rondo) were cultured as described in [24]. Plants from each species were divided into two groups, each of which received different concentrations of N (0.5 to 6.0 mM) in the form of either NO₃^- or NH₄⁺ (applied as Ca(NO₃)₂ or KNO₃ and (NH₄)₂SO₄, respectively). All seeds were surface-sterilised and plants were grown for several days (depending on the plant species) under hydroponic conditions. The pH of the nutrient solutions was buffered with CaCO₃ (5 mM) to pH 6-7, depending on the plant species. The temperature of the solutions was between 18 and 20°C. Nutrient solutions were aerated vigorously (flow rate of 15 mL s^-1) and replaced weekly to minimize the nitrification processes.

Plants were harvested by separating the shoots and roots of each plant. The dry weight of each plant was obtained after drying in an oven at 75-80°C to a constant weight (48-72 h).

ii) Short-term control and ¹⁵N labelling experiments in spinach and pea plants

Spinach seeds (cv. Gigante de Invierno) were germinated and grown hydroponically as described by [26]. N-free Rigaud and Puppo solution [27], which had been diluted (1:2) and modified according to [25] was used during the growth period. The N-free solution was supplemented with 0.5 mM NH₄NO₃ as the only N source for the first 25 days of growth period. Then, spinach plants were fed with a Rigaud and Puppo solution containing 0.5 mM NH₄Cl as the only N source for the last 5 days of the growth period. The pH of the solution was buffered with CaCO₃ (0.25 mM) to pH 6-6.5.

Pea seeds (cv. Sugar-snap) were surface-sterilised according to [28] and then germinated as described in [25]. One-week-old pea seedlings were transferred into tanks (volume: 8 L) in groups of eight and grown in controlled-environment chambers at 275-300 μmol photons m^-2 s^-1, 22/18°C (day/night), 60/70% relative humidity and a 14 h light/10 h dark photoperiod for 1-2 weeks, until the second node stage was reached. The hydroponic vessels contained aerated (0.4 L air min⁻¹ L⁻¹) N-free Rigaud and Puppo solution [27], which had been diluted (1:2) and modified according to [25]. A solution of 0.5 mM NH₄⁺ was supplied as NH₄Cl during the growth period as the only N source. The pH of the solution was buffered with CaCO₃ (2.5 mM) to 7-7.3.

Either spinach or pea plants were then transferred to a solution at pH 6 (KP buffer, 10 mM) or pH 9 (H₃BO₃/NaOH buffer, 50 mM) in a sealed 125-ml Erlenmeyer flask, such that the roots were fully immersed in 100 mL of solution. Fully ¹⁵N-labelled ¹⁵NH₄Cl was injected and rapidly mixed to a final concentration of 10 mM NH₄⁺. Plants from both pH levels were harvested by separating the shoots and roots of each plant at 0, 1, 7.5 (for spinach), 15, 30, 60 and 120 min after the ¹⁵NH₄Cl injection. In order to evaluate how the pH increase affects ion uptake per se, we have used as control a nutrient solution containing RbNO₃ (1 mM), instead of ¹⁵NH₄Cl. This control was performed exclusively on spinach, which is considered a more sensitive species than pea. Internal Rb⁺ and NO₃^- contents were determined in shoots and roots at 7.5, 30 and 120 min after RbNO₃ injection, as tracers of cation and anion uptake respectively in different pHs.

For the uptake experiments, the applied light intensity during the pH and RbNO₃ or ¹⁵N-labelling short-term applications was 750-800 μmol photons m^-2 s^-1 to enhance the absorption process.

pH measurements were determined after the short-term experiments in order to verify that the pH of the solution was properly buffered and that there were no great changes in the pH due to the root ionic exchanges (ion influx/efflux) (Additional file 1).

Isotopic N Composition and N accumulation

Five to eight milligrams of powdered plant material from each sample (shoots and roots) was separately packed in tin capsules. The ¹⁵N/¹⁴N isotope ratios of these samples were determined by isotope ratio mass spectrometry (isoprime isotope ratio mass spectrometer - IRMS, Micromass-GV Instruments, UK). The N isotope composition results are expressed as δ¹⁵N, in parts per thousand (‰) relative to atmospheric N₂: δ¹⁵N (‰) = [(R_sample/R_standard)-1] * 1000, where R_sample is the ¹⁵N/¹⁴N ratio of the sample and R_standard is the ¹⁵N/¹⁴N ratio of the atmospheric N₂. Plant material that had previously been calibrated against a standard material of known isotope composition was used as a working standard for batch calibration during the isotope ratio analyses. The ¹⁵N contents (total, ¹⁵NH₄⁺ and ¹⁵NH₃) were obtained using δ¹⁵N and the total percentage of N for each plant tissue (leaves and roots), and ¹⁵N contents for the external NH₄⁺ and NH₃ were calculated using the Henderson-Hasselbalch equation, which takes into account the external pH. The percentages of NH₃ molecules (relative to the total [NH₄⁺ + NH₃] molecules) at pH 6.08 and pH 9.0 were 0.0676% and 35.993%, respectively (see Additional file 2). Plant tolerance to NH₄⁺ nutrition was calculated as the ratio between biomass accumulation of NH₄⁺- and NO₃^--fed plants at the same N concentration [22]. The δ¹⁵N data corresponding to the N sources used ranged from +0.03 to +2.31 for NH₄⁺ and -1.514 to +0.3 ‰ for NO₃^-.

Determination of inorganic soluble ion content

Plant extracts with soluble ionic contents from shoots and roots were obtained from dry tissues incubated in a bath in 1-2 mL of milli-Q water at 85°C for 10 min, followed by centrifugation (20,000×g, 30 min). The supernatants were stored at -20°C until analysis by ion chromatography. Soluble cation content (Rb⁺) was determined as described in [27] using an isocratic method with 20 mM metanosulphonic acid solution. Soluble anion content (NO₃^-) determination was carried out by the gradient method given by [27]. Rb⁺ content was below the detection limit in shoots.

Statistical analyses

All statistical analyses were performed with Statistical Product and Service Solutions (SPSS) for Windows, version 17.0.

i) Statistical analysis of the natural isotopic abundance experiment in several plant species

We examined results for nine species using analysis of variance to test for effects and interactions of the N treatments (source and concentration) and whether these changed according to the organ and species tested. Organ was included as a factor exclusively in the natural isotopic composition ANOVA test because it was meaningless to include it in the total biomass and total biomass ratio (NH₄⁺/NO₃^-) ANOVA tests.

ii) Statistical analysis for short-term experiments in spinach and pea plants

One-way analysis of variance (ANOVA; factor: time) was performed. The homogeneity of variance was tested using the Levene test [29]. Least significant difference (LSD) statistics were applied for variables with homogeneity of variance, and the Dunnett T3 test [30] was used for cases of non-homoscedasticity. The pHs were compared using Student's t-test for each time point independently, and homoscedasticity was determined using the Levene test [29].

All statistical analyses were conducted at a significance level of 5% (P ≤ 0.05). The results of this study were obtained for plants cultured in several independent series. For the plant species lettuce (cv. Marine), spinach (cv. Spinner), tomato (cv. Trust), pea (cv. Eclipse) and lupin (cv. Albus), plant material from six plants was mixed and analysed in three independent series. For spinach (cv. Gigante de invierno), pea (cv. Sugar-snap and Rondo), carob, perennial ryegrass (cv. Herbus), white clover (cv. Huia) and Acacia sp., at least one sample was analysed for each of three independent series.

Results

Although the δ¹⁵N values of the sources, NO₃^- and NH₄⁺, similarly ranged from -1.514 to +2.31 ‰, the δ¹⁵N observed for several plant species was significantly different when N was provided either as NO₃^- or NH₄⁺ (Table 1). In general, four trends emerged from the natural isotopic signature data (Figure 1): 1) NO₃^--fed plants tended to be enriched in the heavier N isotope, whereas NH₄⁺-fed plants were depleted compared with their respective N sources; 2) for the same external N concentration, the degree of fractionation depended on the plant species; 3) the δ¹⁵N values of shoots and roots were not the same but followed similar patterns; and 4) in contrast to the NO₃^--fed plants, which had δ¹⁵N values that were insensitive to the N concentration, under NH₄⁺ nutrition, fractionation tended to increase with the N concentration within plant species (Table 2). These four trends were supported by the results displayed in Tables 1 and 2 from the analyses of variance of N, species and organ effects. The source of N had a global effect on the isotopic composition (‰) and total biomass (g DW) (Table 1). Moreover, significant two-way interactions between the N source and N concentration (N source × N conc.) and the N source and species (N source × sp.) on the δ¹⁵N and the total biomass were observed (Table 1). Due to the strong effect of the N source on the δ¹⁵N, the main effects of N concentration, species and organ type was analysed in NO₃^-- and NH₄⁺- fed plants separately (Table 2). In NH₄⁺-fed plants, the N concentration, species and organ type had an effect on the natural isotopic abundance; however, in NO₃^-- fed plants, only the diversity (species) factor had an effect on the δ¹⁵N (Table 2).

Table 1 Analysis of variance of the N sources, N concentrations and species.

Figure 1

Natural N isotopic composition of nine plant species with different sensitivity to NH ₄ ⁺ nutrition. Natural isotopic signatures (δ¹⁵N, ‰) of the shoots (A) and roots (B) of several plant species cultured under hydroponic conditions with different concentrations of NH₄⁺ (●) or NO₃^- (○) as the sole N source. The following numbers indicate the species that correspond to each point: (1) Lactuca sativa L., (2) Spinacia oleracea L., (3) Solanum lycopersicum L., (4) Lolium perenne L., (5) Pisum sativum L., (6) Lupinus albus L., (7) Trifolium repens L., (8) Ceratonia siliqua sp., and (9) Acacia aneura sp. Each point is the average of several biological replicates (at least n = 3, depending on the species; see Methods). δ¹⁵N of the N sources: NO₃^- = +0.3 and -1.514 and NH₄⁺ = +0.029, +0.5 and +2.31 ‰.

Table 2 Analysis of variance of the N concentrations, species and organ effects.

Biomass accumulation in NH₄⁺- and NO₃^--fed plants at the same N concentration was dependent on the N concentration in the root medium and on the plant species concerned (Table 2). The degree of the effect of the N concentration on the total plant biomass (growth stimulation with NO₃^- nutrition or growth inhibition with NH₄⁺ nutrition) depended on the species, as shown by the significant interaction of N conc. × sp. for both N sources (Table 2).

The ratio of biomass accumulations between the NH₄⁺- and NO₃^--fed plants was therefore used as an indicator of each plant species' sensitivity (or tolerance) to NH₄⁺ nutrition. The N concentration and diversity also influenced the total biomass ratio of NH₄⁺- and NO₃^--fed plants (Table 2). A very strong correlation between the root δ¹⁵N of NH₄⁺-fed plants and the ratio of biomass accumulation between the NH₄⁺- and NO₃^--fed plants was observed (Figure 2). Thus, the lower biomass ratios (i.e., lower tolerance to NH₄⁺) observed for seven species and cultivars, which presented different degrees of tolerance to NH₄⁺ nutrition grown with several N concentrations, were associated with depletion of the heavier N isotope in the plant material studied (Figure 2). Hence, the most sensitive plants to NH₄⁺ were the most depleted of ¹⁵N (Additional file 3 table S1). The Ceratonia species (carob) showed a unique behaviour relative to the other herbaceous species; its much higher biomass ratios for the negative δ¹⁵N values did not fit within the correlation (see Additional file 3, table S1). The ratio of the whole plant biomass accumulation (NH₄⁺/NO₃^-) in Acacia species was not measured. Hence, they were excluded from the dataset in Figure 2.

Figure 2

Root isotopic signatures (δ¹⁵N, ‰) of NH ₄ ⁺-fed plants correlated with the plant NH ₄ ⁺ toxicity/tolerance indicator (plant biomass ratio NH ₄ ⁺/NO ₃ ^- for each N concentration). The following N concentrations were represented in this analysis: 0.5 mM (upward triangle), 1.5 mM (circle), 2.5 mM (upside down triangle), 3 mM (square), 5 mM (star) and 6 mM (diamond). δ¹⁵N data of the (NH₄)₂SO₄ used in NH₄⁺-fed plants were +0.029, +0.5 and +2.31 ‰, and all three values fall within the area indicated (upper part of graph). The plant species that were cultured hydroponically and used for this statistical analysis were lettuce, spinach, tomato, ryegrass, pea, lupin and white clover. The dataset displayed represents the average values ± SE (at least n = 3, depending on species; see Methods). Linear regression was performed at P ≤ 0.05.

Natural soils rarely exhibit pH values close to the pKa of NH₄⁺ (~ 9.25); therefore, NH₃ is present in very small amounts under normal external pH conditions [2]. In the short-term experiments described herein, three- and four-week-old N-deficient pea and spinach plants, respectively, were transferred to a 100% ¹⁵N-labelled 10 mM NH₄⁺ solution. δ¹⁵N was used as a tool to determine the amount of ¹⁵N that enters the plant roots under the experimental conditions, and a higher increase in the total ¹⁵N content was observed at pH 9 than at pH 6 in both plant species (Figure 3B and 3D). In plants with higher NH₄⁺ sensitivity, i.e., spinach, the ¹⁵NH₃/¹⁵NH₄⁺ absorption reached the asymptotic trend moment in the curve in a shorter period of time than pea plants (Figure 3B and 3D). In shoots, the total ¹⁵N content per DW g was lower in spinach than in pea plants (Figure 3A and 3C). The content of ¹⁵N in spinach shoots was higher in pH 9 than in pH 6 (Figure 3A), whereas in pea plants no difference was observed between pHs during the initial 15 min (Figure 3C). This result indicates that in spinach plants the N is translocated immediately from the roots to the shoot, while in pea plants N translocation is delayed relative to N uptake. At 120 min, opposite effects between pHs were shown in both plant species. In spinach shoots, higher ¹⁵N content was displayed at pH 6, while pea shoots showed higher ¹⁵N content at pH 9 (Figure 3A and 3C). On the other hand, the internal root ¹⁵N content was related to the proportion of NH₄⁺ and NH₃ in the external solution at pH 6 and 9 (Figure 4), as calculated using the Henderson-Hasselbalch equation (see Additional file 2). In both plant species, some important differences were found between the plants at pH 6 and 9 in terms of the proportion of ¹⁵N uptake from the external NH₄⁺ source during the initial 15 min after transfer to a different pH (Figure 4A and 4C), whereas the uptake rates of ¹⁵N from the external NH₄⁺ were similar at both pH levels 60 min after the beginning of the experiment (Figure 4A and 4C). The most remarkable finding, however, was a drastic increase in ¹⁵N uptake from the external NH₃ source at pH 9, which was maintained throughout the experiment (up to 120 min, Figure 4B and 4D).

Figure 3

¹⁵N contents in tissues of spinach and pea plants. ¹⁵N content (μmol g^-1 DW) calculated from the δ¹⁵N data, in shoots (A and C) and roots (B and D) of spinach (A and B) and pea (C and D) plants transferred from pH 7 to pH 6 (○) or pH 9 (●).

Figure 4

Root ¹⁵NH ₄ ⁺ and ¹⁵NH ₃ contents calculated from the total ¹⁵N uptake. ¹⁵N content accumulated from ¹⁵NH₄⁺ absorption (μmol g^-1 DW) in spinach (A) and pea (C) plants. ¹⁵N content accumulated from ¹⁵NH₃ absorption (μmol g^-1 DW) in spinach (B) and pea (D) plants. (B1 and D1) Magnified portions of plots (B and D respectively) showing the ¹⁵N content that accumulated as a result of external ¹⁵NH₃ absorption at pH 6 (μmol g^-1 DW). The partitioning between NH₃ and NH₄⁺ has been calculated using the Henderson-Hasselbalch equation (see Additional file 2). Data represent the average values ± SE (n = 3). Letters represent significant differences (P ≤ 0.05) during exposure to pH 6 (A, B, C and D) and pH 9 (a, b, c and d). An asterisk (*) denotes significant differences between pH 6 and 9 (P ≤ 0.05).

On the other hand, a broad range of K⁺ channels have been shown to allow significant levels of NH₄⁺ to permeate [31], and at the same time Rb⁺ is commonly used as a K⁺ analogue in physiological studies [32], as its size and permeability characteristics are very similar to those of K⁺[33]. Thus we have used Rb⁺ as a tracer for evaluating the effect of pH increase in cation uptake. The uptake rates of Rb⁺ from the external RbNO₃ source were similar at both pH levels throughout the experiment (Figure 5A). The anion (NO₃^-) absorption was lower under alkaline than acidic conditions (Figure 5B). In shoots, the internal NO₃^- contents were similar in both external pHs (not shown). Therefore, all the effects observed in this study under NH₄⁺ nutrition and different pH conditions (Figures 3 and 4) can be just attributed to the ratio between NH₃ and NH₄⁺.

Figure 5

Root ion contents of spinach plants. Root ion content (μmol g^-1 DW) of plants transferred from pH 7 to pH 6 (○) or pH 9 (●). (A) Rb⁺ content. (B) NO₃^- content.

Discussion

Natural isotopic abundances of N in plants grown with NO₃^- or NH₄⁺

An important degree of fractionation, determined as the difference between the δ¹⁵N of the N source and that of the plant, was observed when plants were grown hydroponically with a known concentration of a single N form in a controlled environment (Figure 1). Thus, NO₃^-- fed plants tended to be enriched in the heavier N isotope in relation to the source, whereas NH₄⁺-fed plants tended to be depleted (Figure 1).

The degree of fractionation in the reaction rates of the two N isotopes (¹⁴N and ¹⁵N) reflects both their mass differences and the force constants of the bonds they form. A significant isotope effect due to ionisation would therefore not be expected [34].

The positive δ¹⁵N values for NO₃^--fed plants may be associated with N loss from the plant in the form of root efflux and exudates [6, 7, 35] or loss of NH₃ through the stomata [36–39], which favours the lighter isotope [40]. The ratio between the root and shoot δ¹⁵N values may also depend on the partitioning of N metabolism between the roots and shoots. The isotopic effect for nitrate reductase enzyme is 1.015 (or higher, see [4] and references therein) and that associated with glutamine synthetase is 1.017 [41]; therefore, the resulting organic compounds (amino acids) would therefore be depleted of ¹⁵N in relation to the inorganic N pool. Thus, depending on the main site, shoots or roots, of N reduction and assimilation, the tissues would present distinct δ¹⁵N values. Since NO₃^- and NH₄⁺ are not major constituents of the phloem, most of the N translocated into the plant in the organic form is likely to be depleted of ¹⁵N compared with N source. Because the main site of NO₃^- reduction for each species is dependent on the N status of the plant, the relationship between the δ¹⁵N of roots and shoots may vary for the same plant species according to the external N availability and for the same external conditions according to plant species (Figure 1) and phenological stage. Thus, under NO₃^- nutrition, there was no significant effect of the organ on the natural isotopic abundance of N (Table 2).

In contrast, the shoots of NH₄⁺-fed plants were significantly enriched in ¹⁵N (Table 2) relative to the roots (see Additional file 3, tables S2 and S3). Among the various external factors, the source and concentration of N have an effect on stomatal NH₃ emissions [36, 37]. Thus, losses of NH₃ from the stomata take place in NH₄⁺-fed plants at high N concentrations [38, 39]. This process will favour the lighter isotope emission and enrich the plant tissue (leaf specially) in ¹⁵N because the isotopic effect of NH₃ (aq) exchange with NH₃ (g) has been estimated to be 1.005. In other words, NH₃ (g) is enriched in ¹⁴N by ~ 5 ‰ relative to NH₃ (aq) [21]. In agreement with this reasoning, the nitrogen isotopic fractionation against ¹⁵N caused by volatilisation of NH₃ has been shown in the aerial part of wheat plants [40]. Hence, in light of the N dynamics inside the plant, it is difficult to explain how the whole NH₄⁺-fed plants can be depleted of the heavier N isotope.

N Isotopic fractionation and NH₄⁺toxicity mechanisms

Some studies have examined isotopic fractionation in plants grown with NH₄⁺ nutrition under LATS controlled conditions, and contrasting results were obtained. For instance, isotopic fractionation in NH₄⁺-fed (4.6 mM) Pinus sylvestris ranged from 0.9 to 5.8 [42]. For Oryza sativa L., the fractionation was dependent on the external NH₄⁺ concentration, which ranged from -7.8 to -18 ‰ when the external NH₄⁺ concentrations ranged from 0.4 to 7.2 mM [7]. In agreement with this latter trend in rice, our results showed that the fractionation tended to increase with the N concentration for most of the plant species studied under NH₄⁺ nutrition (Figure 1, Table 2 and Additional file 3, tables S2 and S3). Hence, the organ δ¹⁵N values were closer to the source δ¹⁵N in low N availability conditions (at low N concentrations) for NH₄⁺-fed plants [6] (Figure 1). Likewise, if the N concentration increases, the amount of substrate becomes unlimited and the isotope effect is observed [6] (Figure 1). However, the δ¹⁵N values from NO₃^--fed plants were almost insensitive to the N concentration (Figure 1 and Table 2), which agrees with experiments in rice [7]. Thus, even if organic N compounds were lost, this phenomenon would not be sufficient to explain the plant depletion of ¹⁵N as the assimilatory enzymes discriminate against the heavier N isotope [4].

If we consider the mechanisms of NH₄⁺ toxicity, a recent study examined the causes of the primary root growth suppression by NH₄⁺ nutrition [43]. It demonstrated that the NH₄⁺-mediated inhibition of primary root growth is mostly due to a repression of cell elongation rather than cell division inhibition. Moreover, these authors linked this phenomenon to two mechanisms of NH₄⁺ toxicity [44–46]. First, the futile plasma transmembrane cycle of NH₄⁺ uptake and efflux through cell roots, with the subsequent high energetic cost, might explain the different tolerances exhibited by different plant species when NH₄⁺ is supplied at high concentrations [44]. Hence, Li et al. [43] showed that NH₄⁺ efflux is induced by high NH₄⁺ concentrations in the Arabidopsis root elongation zone, which coincides with the inhibitory effect of NH₄⁺ on cell length and primary root elongation. They also associated the NH₄⁺-induced efflux in the root elongation zone with the enzyme GDP-mannose pyrophosphorylase (GMPase). The implication of GMPase in the NH₄⁺ sensitivity of Arabidopsis roots represents the second (and last) mechanism of NH₄⁺ toxicity [45, 46]. Therefore, Li et al. pointed out that GMPase regulates the process of root NH₄⁺ efflux, and showed that GMPase mutants had a higher net NH₄⁺ efflux (1.8 fold) in the root elongation zone relative to wild-type Arabidopsis plants [43].

In our study, we did not determine the net NH₄⁺ fluxes, but previous findings demonstrated that the root NH₄⁺-induced efflux occurs in a broad range of plant species and are more or less significant depending on the NH₄⁺ sensitivity of the plant species [44]. So, the mechanism of NH₄⁺ ejection from the root cell, if it occurred, would significantly contribute towards the global ¹⁵N depletion of the NH₄⁺-fed plants through a discriminatory mechanism against the lighter N isotope (i.e., favouring the ¹⁵N isotope). However, the fractionation mechanism against ¹⁴N is a thermodynamically unlikely event due to the differences in the physical and chemical properties of isotopic compounds. Thus, the heavier molecules have a lower diffusion velocity, and generally, the heavier molecules have higher binding energies [47].

Furthermore, the relative abundances of the stable isotopes in living organisms depend on the isotopic composition of their food sources and their internal fractionation processes [48]. Thus, taking into account the development of the relative abundance of the stable isotopes across the food web, internal fractionation generally leads to an enrichment of the heavier isotope in consumers relative to their diet [48]. The negative values for the natural isotopic fractionation observed in NH₄⁺-fed plants must therefore be related to the chemical properties of the NH₄⁺ ion in solution and the NH₄⁺/NH₃-uptake mechanisms. When NH₄⁺ is applied as the only N source, the NH₄⁺ and NH₃ forms are present in the nutrient solution. However, these molecular and ionic forms do not have exactly the same natural isotopic signatures because there is a 1.020 thermodynamic isotope effect between NH₃ (aq) and NH₄⁺ (aq), such that NH₃ (aq) is depleted of ¹⁵N by 20 ‰ relative to NH₄⁺ (aq) [20]. To interpret the negative values of the whole plant δ¹⁵N, we hypothesise that a portion of the N enters the root as NH₃, which leads to the depletion of the heavier isotope in the plant.

A proposal that relates N isotopic fractionation and NH₄⁺toxicity mechanism

When the whole plant is considered and NH₄⁺ is the only available N source, the isotopic N signature of the plant would therefore be related to the amount of NH₃ transported. Using the ratio between the biomass accumulations of NH₄⁺- and NO₃^--fed plants as an indicator of NH₄⁺ tolerance [22], we can relate NH₄⁺ tolerance to the root δ¹⁵N of NH₄⁺-fed plants. Plants that were less tolerant to NH₄⁺ nutrition were the most depleted of the heavier isotope (Figure 2; Additional file 3, table S1), and presumably the uptake of NH₃ was more important in those plants. According to our hypothesis, lettuce, spinach and tomato were the most sensitive to NH₄⁺ nutrition of the plant species studied (Figure 2 and Additional file 3 table S1). Moreover, the "plant sensitivity to NH₄⁺ nutrition" variable, expressed as the ratio of the biomasses of NH₄⁺/NO₃^--fed plants, can explain 69% of the root δ¹⁵N variation observed in the dataset (Figure 2). Hence, although the fraction of NH₃ in solution at pH 6-7 is very small (approx. 0.07-0.6%), the transient alkalinisation of the cytosol reported after NH₃ uptake can be attributed to rapid diffusion of NH₃ across the plasma membrane and its subsequent protonation within the cytosol [49, 50]. The increased NH₃ concentration will therefore consume the established Δ μ_H+, thereby contributing to a higher energetic cost to balance it. This may also be related to membrane depolarisation events observed after NH₄⁺ application in NH₄⁺-tolerant plants or to the higher energetic burden reportedly required to maintain membrane potentials in NH₄⁺-sensitive species [44].

In order to test the viability of our hypothesis, short-term experiments were performed using two plant species that showed different tolerance to NH₄⁺ nutrition at two pHs; a slightly acidic one pH (6.0), and an alkaline pH (9.0) which favoured the neutral form (NH₃). Spinach (sensitive; Figure 2) and pea (tolerant; Figure 2) receiving ¹⁵NH₄⁺ as the only N source showed that 2 h was sufficient to demonstrate that N uptake was faster in plants transferred from pH 6-7 to pH 9 than in those transferred from pH 6-7 to pH 6 (Figure 3B and 3C). The differences shown in shoot ¹⁵N contents between pHs and species (Figure 3A and 3C) suggest interesting dissimilarities in uptake and transport systems, linked to the degree of sensitivity/tolerance of these species to NH₄⁺. This finding may be related to the different distribution of incorporated NH₄⁺ reported in both species (shoot in spinach and root in pea plants) [51]. In this work it is proposed that differences in the site of NH₄⁺ assimilation is linked to NH₄⁺ tolerance. On the other hand, taking into consideration the N absorbed by the plants and the dissociation constant of the ionic form, most of the difference in N uptake at pH 6 and pH 9 is likely related to a higher proportion of NH₃ under alkaline conditions (Figure 4B and 4D). These observations are consistent with the hypothesis that the NH₃ form is involved in the uptake of reduced N by the cell in the LATS activity range.

Physiological studies have indicated that transport of NH₃ across membranes occurs and may become significant at high NH₄⁺ concentrations or at high pHs [16]. Indeed, NH₃ transport has been described as a function of the HATS in Escherichia coli [52, 53]. The first hints of protein involvement in plant NH₃ transport came from nodules of legume rhizobia symbiosis and restoration of NH₃ transport in yeast mutants complemented with three aquaporins from wheat roots. This complementation was found to be pH-dependent, with progressively better growth being observed at increasing pH, and was thus indicative of transport of neutral NH₃ rather than charged NH₄⁺[54]. Recently, the transport of NH₃, rather than NH₄⁺, by the AtAMT2 transporter was also shown [14, 15]. Furthermore, the incubation of an illuminated suspension of mesophyll cell protoplasts from Digitaria sanguinalis, which had been preloaded with a pH-specific fluorescent probe, with 20 mM of NH₄Cl showed rapid alkalinisation of the cytosolic pH [55], which may be explained on the basis of NH₃ uptake. Further examples of transient alkalinisation of the cytosol have been reported in root hair cells of rice and maize after the addition of 2 mM NH₄⁺ to a previously N-free bathing solution [50], which indicates that NH₃ permeates cells [50, 55]. This process will contribute to consumption of the established Δ μH⁺ and agrees with the hypothesis that the toxic effect of NH₃ is associated with intracellular pH changes [44]. All of these studies together demonstrate that NH₄⁺ may permeate cells in its neutral form (NH₃) and therefore tends to increase cytosolic pH.

The level of GMPase activity has been proposed to be a key factor in the regulation of Arabidopsis sensitivity to NH₄⁺[45]. Interestingly, these authors showed that GMPase activity is seemingly regulated by pH. Using in vitro experiments with recombinant wild-type and GMPase mutant proteins, GMPase activity was decreased by alkaline pH. In plants cultured on NO₃^-, a considerable decrease in GMPase activity was observed with increasing pHs from 5.7 to 6.7 of the plant growth medium. Moreover, plants grown in the presence of NH₄⁺ showed lower GMPase activities relative to that shown by NO₃^--fed plants at the same external pH [45]. This could indicate that the transient cytosolic alkalinisation previously reported in NH₄⁺ uptake (reviewed in [56]) may trigger the decrease of GMPase activity stimulated by NH₄⁺ provision [45]. In fact, Qin et al. have hypothesised that this cytosolic alkalinisation may play a role in the inhibition of GMPase activity by NH₄⁺[45].

Thus, in view of our results and these previous findings, we propose the existence of a mechanism that recruited the NH₄⁺ in the molecular form (NH₃) under LATS conditions, which would cause in parallel depletion in the heavier N isotope, as well as an alkalinisation of cytosol in root cells. It would trigger a decrease in GMPase activity and the subsequent downstream molecular events, i.e., deficiencies in protein N-glycosylation, the unfolded protein response and cell death in the roots [45], which are important for the inhibition of Arabidopsis growth by NH₄⁺ application [45]. Moreover, reductions in cellulose biosynthesis, cell wall stability and cell viability shown in a null mutant of GMPase (cty1-2) are the result of an N-glycosylation deficiency [57]. The disturbance of cell wall biosynthesis caused by the decreased GMPase activity under NH₄⁺ nutrition and the subsequent protein N-glycosylation deficiency [45] has been related to the NH₄⁺ flux [43]. Our proposal, therefore, is compatible with the two related NH₄⁺-toxicity mechanisms [43] proposed by Britto et al. [44] and Qin et al. [45].

On the other hand, several reports have suggested that K⁺ channels are an important component of the LATS for NH₄⁺[58]. It has been shown that NH₄⁺ produces similar, but weaker, currents compared to K⁺ in intact root cells or in protoplasts ([10] and references therein) and that a single amino acid substitution in a K⁺ channel can dramatically increase NH₄⁺ permeability [59]. Indeed, a broad range of K⁺ channels have been shown to be permeable to NH₄⁺[8, 60], and most allow significant levels of NH₄⁺ to permeate [31]. Alternatively, it might be expected that some channels and transporters poorly distinguish between K⁺ and NH₄⁺. In fact, it has been shown that the futile NH₄⁺ cycling, which was shown in NH₄⁺-sensitive plants under NH₄⁺ nutrition [44], is alleviated by elevated K⁺ levels and that low-affinity NH₄⁺ transport is mediated by two components, one of which is K⁺ sensitive and the other is K⁺ independent [31]. As NH₄⁺ transport through K⁺ channels would be in the ionic form, no ¹⁵N fractionation is expected to be associated with it.

Conclusions

Based on the results presented herein, we show that plants fed with NH₄⁺ as the sole source of N are depleted of ¹⁵N in a concentration-dependent manner. We have observed a relationship between ¹⁴N/¹⁵N fractionation and the sensitivity of plants to NH₄⁺ nutrition. We show that the most sensitive plants have the most negative δ¹⁵N values. Moreover, our data of ¹⁵N uptake at pH 6.0 and 9.0 together with other data found in the literature indicate that part of N uptake by the plant may occurs as NH₃. Accordingly, current data has suggested that the LATS for NH₄⁺ has at least two components. One component is involved in the transport of NH₃ and would therefore indirectly discriminate against the heaviest N stable isotope due to the balance between ionic and molecular forms in the nutrient solution. This transport mechanism could correspond to the K⁺-independent component of NH₄⁺ transport suggested previously [31]. The second component would be an NH₄⁺-specific transport system, which interferes with K⁺ transport and does not discriminate against ¹⁵N. We propose that the negative values of δ¹⁵N observed in hydroponically grown plants are related to this NH₃ uptake, which imprints a permanent N signature (δ¹⁵N) under steady-state external N conditions and contributes to the current understanding of the origin of NH₄⁺ toxicity.