Plant Physiol. (1998) 118: 1361-1368

Regulation of Apoplastic NH₄⁺ Concentration in Leaves of Oilseed Rape¹

Kent Høier Nielsen^* and Jan Kofod Schjoerring

Plant Nutrition Laboratory, Department of Agricultural Sciences, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

Regulation of apoplastic NH₄⁺ concentration in leaves of oilseed rape (Brassica napus L.) was studied using a vacuum-infiltration technique that allowed controlled manipulations of the apoplastic solution. In leaves infiltrated with NH₄⁺-free solution, the apoplastic NH₄⁺ concentration returned in less than 1.5 min to the preinfiltration level of 0.8 mM. Infiltrated ¹⁵NH₄⁺ was rapidly diluted by ¹⁴NH₄⁺/¹⁴NH₃ effluxed from the cell. The exchange rate of ¹⁵N/¹⁴N over the apoplast due to combined ¹⁴N efflux from the symplast and ¹⁵N influx from the apoplastic solution was 29.4 µmol g¹ fresh weight h¹ between 0 and 5 min after infiltration. The net uptake of NH₄⁺ into the leaf cells increased linearly with apoplastic NH₄⁺ concentrations between 2 and 10 mM and could be partially inhibited by the channel inhibitors La³⁺ and tetraethylammonium and by Na⁺ and K⁺. When apoplastic pH increased from 5.0 to 8.0, the steady-state apoplastic NH₄⁺ concentration decreased from 1.0 to 0.3 mM. Increasing temperature increased the rate of NH₄⁺ net uptake and reduced the apoplastic steady-state NH₄⁺ concentration. We conclude that the apoplastic solution in leaves of oilseed rape constitutes a highly dynamic NH₄⁺ pool.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

NH₄⁺ is constantly generated in large quantities in plant leaves by processes such as photorespiration, nitrate reduction, protein turnover, and lignin biosynthesis (Joy, 1988; Leegood et al., 1995). Refixation of NH₄⁺ takes place mainly in the chloroplasts and is catalyzed by the chloroplastic isoform of Gln synthetase, GS₂ (Leegood et al., 1995). In addition to being a central metabolic intermediate, NH₄⁺ may be translocated to the leaves from the roots (Cramer and Lewis, 1993; Mattsson and Schjoerring, 1996).

The rapid turnover of NH₄⁺ in plant leaves leads to the establishment of a finite NH₄⁺ concentration in the leaf apoplastic solution (Husted and Schjoerring, 1995). This concentration and the concentration of H⁺ determines the size of the NH₃ compensation point (i.e. the NH₃ mole fraction in the air within the substomatal cavities; Farquhar et al., 1980; Husted and Schjoerring, 1996). The NH₃ compensation point ranges between 0.1 and 20 nmol mol¹ air and is thus of the same order of magnitude as the naturally occurring atmospheric NH₃ concentration (Sutton et al., 1994). At an NH₃ compensation point of 5 nmol mol¹, for example, this would under conditions of equilibrium correspond to an apoplastic NH₄⁺ concentration of 1 mM at 20°C and pH 5.8 (Husted and Schjoerring, 1996). The existence of an NH₃ compensation point implies that vegetation has a major influence on the transport and budgets of atmospheric NH₃, a pollutant with damaging environmental impacts (Langford and Fehsenfeld, 1992; Dentener and Crutzen, 1994; Sutton et al., 1995).

The concentration of NH₄⁺ in the leaf apoplastic solution is very sensitive to leaf N status and external N supply. Therefore, the apoplastic NH₄⁺ concentration may be about 10 times higher in oilseed rape (Brassica napus L.) plant leaves treated with high N than in leaves treated with low N (Husted and Schjoerring, 1996). Barley plants having access to NH₄⁺ in the root medium have higher apoplastic NH₄⁺ concentrations than plants absorbing NO₃, and the leaf apoplastic NH₄⁺ concentration increases with the NH₄⁺ concentration in the root medium (Mattsson and Schjoerring, 1996). Inhibition of Gln synthetase leads to a rapid and very substantial increase in apoplastic NH₄⁺ (Husted and Schjoerring, 1995), and barley mutants with reduced Gln synthetase activity have increased apoplastic NH₄⁺ relative to wild-type plants (Mattsson et al., 1997).

Despite the importance of leaf apoplastic NH₄⁺ concentration in NH₄⁺ recovery and plant-atmosphere NH₃ exchange, very little information is available concerning the transport of NH₄⁺ between the leaf apoplast and symplast. In leaf discs of bean, Raven and Farquhar (1981) observed that uptake of methylammonium (an NH₄⁺ analog) could not be accounted for by passive diffusion but seemed to be mediated by some kind of energy-requiring transport system. In roots of various plant species as well as in Chara corallina, a high-affinity transport system showing Michaelis-Menten kinetics with a K_m of approximately 15 to 40 µM and a low-affinity transport system showing a linear response to external NH₄⁺ have been demonstrated (Ritchie, 1987; Glass et al., 1997). Considering the relatively high concentrations of NH₄⁺ (0.5-1.5 mM) frequently encountered in the leaf apoplastic solution of oilseed rape plants, the low-affinity system appears to be central in NH₄⁺ transport. In roots the low-affinity transport system has been proposed to be a uniport, with fluxes driven by the electrochemical gradient across the plasma membrane (Wang et al., 1994). This uniport may be a specific NH₄⁺ channel, a K⁺ channel, or a shared cation channel (e.g. a K⁺/NH₄⁺ channel, as indicated by Avery et al. [1992] and Schachtman et al. [1992]). Ninnemann et al. (1994) isolated and characterized a gene for a high-affinity NH₄⁺ transporter that was highly expressed in both roots and leaves of Arabidopsis.

A further complicating factor concerning NH₄⁺ transport in leaves relative to that in roots is the possible existence of a large efflux component due to diffusion of dissolved NH₃. Even under conditions in which the intracellular NH₄⁺ concentration is 10 to 100 times lower than the extracellular concentration, a high pH in the cytoplasm (7.0-7.5; Martin et al., 1982) and in the chloroplasts (approximately 8.0 in light) relative to that in the apoplastic solution (approximately 6.0) may maintain a gradient of dissolved NH₃ directed toward the apoplast.

The objective of the present work was to investigate the response of NH₄⁺ transport between the apoplast and symplast of leaf cells in oilseed rape to variations in apoplastic NH₄⁺ concentration. The hypothesis tested was that the apoplastic NH₄⁺ concentration is highly regulated and rapidly attains a steady-state level under changing conditions. A vacuum-infiltration method was used to manipulate the apoplastic NH₄⁺ concentration and to introduce various transport inhibitors into the apoplast. Effects of controlled changes in different parameters such as temperature and pH on the steady-state NH₄⁺ concentration and NH₄⁺ net transport were elucidated. Finally, the stable isotope ¹⁵N was used to assess the contribution of bidirectional NH₃/NH₄⁺ transport over the plasma membrane to the maintenance of apoplastic NH₄⁺ homeostasis.

	MATERIALS AND METHODS

Top Abstract Introduction Methods Results Discussion References

Seeds of oilseed rape (Brassica napus L. cv Global) were germinated in the dark on wet filter paper for 4 d prior to planting in 0.0025-m³ self-watering pots (four plants per pot). The pots were filled with a growth medium consisting of a 1:1 mixture of soil to sand and containing 0.15 mol NH₄NO₃ per pot, supplemented with additional nutrients as described by Husted and Schjoerring (1995). Plants were grown in a greenhouse at a day/night temperature cycle of approximately 18°C/14°C (70% ± 5% RH) under a 16-h photoperiod with a PPFD > 400 µmol m² s¹.

Before experiments, fully developed green leaves were cut off at the stem and their petioles were cut with a sharp blade under deionized water. Leaves were thereafter transferred to a growth chamber 1 h before experiments to allow adjustment to the environmental conditions under which the experiments were later carried out. Unless otherwise specified, the growth chamber had 70% ± 5% RH, a temperature of 20°C ± 1°C, and a PPFD of 475 ± 5 µmol m² s¹.

Extraction and Analysis of Apoplastic Solution

Apoplastic air volume was determined by infiltrating the leaf disc with a high-viscosity silicone fluid (polydimethylsiloxane: viscosity, 5 centistoke, density, 0.904 g cm³; Dow Corning, Poole, UK). The air volume was calculated as the increase in weight of the leaf disc after infiltration, corrected for the density of the silicone oil. The fraction of leaf apoplastic solution in the extracellular space of the leaf disc was determined by infiltrating the apoplast with indigo carmine (50 µM indigo-5,5disulfonic acid, Sigma) dissolved in 50 mM phosphate buffer at pH 6.2 and adjusted to 350 mosmol with sorbitol. After the dye had infiltrated, the apoplastic solution was isolated by centrifugation, and the dilution of the indigo carmine solution was determined spectrophotometrically at 610 nm.

Cation concentrations in the apoplastic extracts were measured by isocratic HPLC using an IC-Pak C column (Waters-Millipore) at 30°C with a flow rate of 1.0 mL min¹ and an eluent containing 0.1 mM EDTA, 2.5 mM 18-crown-6-ether (Sigma), and 4.0 mM HNO₃.

Measurements of pH in apoplastic extracts were conducted in a microcentrifuge tube using a microelectrode (Smart ISFET Micro Probe, Beckman).

The osmolality of solutions used for infiltration was measured on a cryoscopic osmometer (Osmomat 030, Gonotec, Berlin, Germany). All solutions were prepared from ultrapure water (Milli-Q, Millipore) with an 18.2 m resistance.

Apoplastic NH₄⁺ Homeostasis

To assess the bidirectional transport of NH₃/NH₄⁺ over the plasma membrane, leaf discs were infiltrated with 1 mM ¹⁵NH₄Cl (98% ¹⁵N) in an unbuffered solution containing 280 mM sorbitol (350 mosmol). The leaf discs were then incubated for 5, 10, 15, or 25 min before extraction of the apoplastic solution. During the incubation period the leaf discs were placed on a soaked piece of filter paper in a zippered plastic bag to avoid evaporation of leaf water. The plastic bags were placed in a growth chamber (same conditions as described above).

The ¹⁵N abundance in the extracted apoplastic solution was determined by the Dumas combustion method in a system consisting of an elemental analyzer (ANCA-SL, Europa Scientific, Crewe, UK) coupled to a mass spectrometer (20-20 tracer, Europa Scientific). Because of the low quantity of apoplastic solution obtained from the leaf disc, it was necessary to spike the samples with 10 µg of N in the form of (NH₄)₂SO₄ to facilitate analysis within the detection limit of the mass spectrometer. Samples and spiking solution were mixed in tin capsules and freeze-dried prior to analysis.

NH₄⁺ Net Transport at Different NH₄⁺ Concentrations in the Apoplastic Solution

Influence of Inhibitors on NH₄⁺ Net Uptake

The influence of the two channel blockers, La³⁺ and TEA-Cl, on the net uptake of NH₄⁺ was tested by infiltration with 150 mM Mes buffer, pH 6.2, containing either 10 mM NH₄Cl and 10 mM LaCl₃ or 10 mM NH₄Cl and 10 mM TEA-Cl. Control leaves were infiltrated with 10 mM NH₄Cl only. Leaves were not pretreated with the inhibitors.

Effects of K⁺ and Na⁺ on NH₄⁺ Net Uptake

Influence of pH and Net H⁺ Transport between Apoplast and Symplast on Steady-State NH₄⁺ Concentration in the Apoplastic Solution

The net H⁺ transport during the period of infiltration was determined by measuring the pH in the infiltration solution before and after infiltration (maximum difference 0.4 pH unit; Fig. 5b). The amount of protons required to cause this pH change was subsequently quantified by titration of 100 mL of the buffered infiltration solution, corrected for the dilution with solution already present in the apoplast before the infiltration. The buffer capacity of the latter solution was negligible relative to that of the infiltration buffer solution (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of apoplastic pH on apoplastic NH4+ concentration and H+ net uptake from the apoplastic solution in leaf discs of oilseed rape. a, Steady-state apoplastic NH4+ concentration at different pH values in the apoplastic solution. Leaf discs were infiltrated for 4 min with 150 mM Mes buffer, pH 5.0 and 6.0, or 150 mM Tes buffer, pH 7.0 and 8.0, adjusted to an osmotic potential of 350 mosmol with sorbitol. b, H+ net uptake from the apoplastic solution during the infiltrations described in a. , pH in the infiltration solution prior to infiltration; , pH in the solution extracted after 4 min. Values are the means ± SE of four replicates. FW, Fresh weight.

Effect of Temperature on Steady-State Apoplastic and Symplastic NH₄⁺ Concentrations and Net NH₄⁺ Transport between Apoplast and Symplast

2

1

Calculations

(1)

1

To obtain the net uptake of NH₄⁺ over the plasma membrane per unit leaf fresh weight, the volume of extracellular space (V_apo, L g¹), including both apoplastic water and air, was calculated as:

(2)

where V_air is the volume fraction (L g¹) of the extracellular air space. Finally, the net uptake (FNH₄⁺) was calculated following the equation:

(3)

where C_ex is the molar NH₄⁺ concentration in the apoplastic solution at the conclusion of the experiment and t is the duration in hours of the experimental period. The experimental period was defined as starting at infiltration and ending at the start of centrifugation. The shortest possible duration of the experimental period was 1.5 min, which corresponds to the minimum time required to infiltrate and transfer the leaf disc to the centrifuge.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

The volume of apoplastic air in the leaves ranged from 0.20 to 0.25 mL g¹ fresh weight. The corresponding range for apoplastic water was 0.06 to 0.10 mL g¹ fresh weight. The apoplastic NH₄⁺ concentration in newly sampled leaves was approximately 0.8 mM.

NH₄⁺ Homeostasis in Leaf Apoplastic Solution

View larger version (15K): [in this window] [in a new window]   Figure 1. Time course of apoplastic NH4+ concentration in leaf discs of oilseed rape infiltrated at 20°C with either 0 mM () or 0.8 mM () NH4Cl in a 150 mM Mes buffer solution, pH 6.2, adjusted to 350 mosmol with sorbitol. The concentration at time 0 after infiltration with 0 mM NH4Cl was corrected for the NH4+ already present in the apoplast. Values are the means ± SE of four replicates.

Introduction of a solution containing 1.0 mM ¹⁵N-enriched NH₄⁺ into the apoplast, resulting in an initial ¹⁵N excess of 72 atom % in the apoplastic NH₄⁺ pool, was followed by a very rapid dilution of the ¹⁵N with ¹⁴N (Fig. 2). After 5 min the ¹⁵N excess was reduced to 20.0 atom %, and after 25 min it was reduced to 8.4 atom %. During the same period the apoplastic NH₄⁺ concentration remained constant at 0.8 mM (Fig. 2), and total apoplastic N, including all organic and inorganic N compounds, remained at 7.1 mM (data not shown). In the time intervals 0 to 5, 5 to 10, 10 to 15, and 15 to 25 min after infiltration, the decline in ¹⁵N atom % excess amounted to 75%, 41%, 18%, and 12%, respectively, when expressed relative to the ¹⁵N excess at the start of each period. The corresponding exchange rate of ¹⁵N/¹⁴N over the apoplast due to the combined ¹⁴N efflux from the symplast and ¹⁵N uptake from the apoplastic solution was 29.4 µmol g¹ fresh weight h¹ between 0 and 5 min after infiltration and 3.9 ± 0.7 µmol g¹ fresh weight h¹ between 15 and 25 min after infiltration (Table I).

View larger version (15K): [in this window] [in a new window]   Figure 2. Time course of 15N excess () and NH4+ concentration () in the apoplastic solution of leaf discs of oilseed rape infiltrated with 1.0 mM 15NH4Cl solution adjusted to an osmotic potential of 350 mosmol with sorbitol. Values are the means ± SE of six replicates.

Response of NH₄⁺ Net Uptake to Increasing Apoplastic NH₄⁺ Concentration

View larger version (13K): [in this window] [in a new window]   Figure 3. Net uptake of NH4+ from the apoplastic solution in leaf discs of oilseed rape infiltrated for 4 min at 20°C with 150 mM Mes buffer solutions, pH 6.2, adjusted to an osmotic potential of 350 mosmol with sorbitol and different concentrations of NH4Cl. Values are the means ± SE of four replicates. FW, Fresh weight.

Effect of Inhibitors and Competing Cations on NH₄⁺ Net Uptake

View larger version (44K): [in this window] [in a new window]   Figure 4. Effect of different inhibitors on NH4+ uptake from the apoplastic solution in leaf discs of oilseed rape. a, 20 µM CCCP (a protonophore) and 100 µM DES (an ATPase inhibitor) supplied by petiole feeding during a 1-h pretreatment period and subsequently added to the infiltration solution. b, KCl and NaCl added in increasing concentrations to the infiltration solution. c, 10 mM of the nonspecific channel blocker La3+ (LaCl3). d, 10 mM specific K+-channel blocker TEA-Cl. In addition to the specified inhibitor the infiltration solution contained 150 mM Mes, pH 6.2, and 10 mM NH4Cl and was adjusted to an osmotic potential of 350 mosmol with sorbitol. The experiments were carried out at 20°C with a incubation period of 4 min. Values are the means ± SE of four replicates.

Increasing the concentrations of K⁺ or Na⁺ resulted in a decrease in NH₄⁺ net uptake (Fig. 4b). The inhibition caused by K⁺ was 50% at 100 mM KCl in the infiltration solution. A similar concentration of NaCl resulted in only a 20% decline of NH₄⁺ net uptake.

Effect of Apoplastic pH on Steady-State Apoplastic NH₄⁺ Concentration and Proton Flux

Apoplastic pH also affected the net transport of H⁺ between the apoplast and the symplast (Fig. 5b). At approximately pH 6.5 the net transport of H⁺ was zero. Below this pH value, the leaf cells had a net uptake of H⁺, whereas at higher pH values the net H⁺ transport was in the opposite direction. The magnitude of the net H⁺ transport was 20.4 nmol g¹ fresh weight h¹ at pH 5.0 and 120 nmol g¹ fresh weight h¹ at pH 8.0 (Fig. 5b).

Effect of Temperature on NH₄⁺ Net Uptake and Apoplastic NH₄⁺ Concentration

View larger version (16K): [in this window] [in a new window]   Figure 6. The temperature response of NH4+ net uptake from the apoplastic solution (a), steady-state apoplastic NH4+ concentration (b), and tissue-water NH4+ concentration in leaf discs of oilseed rape (c). Leaf discs were infiltrated for 4 min at the specified temperature with 150 mM Mes buffer, pH 6.2, containing 10 mM NH4Cl and adjusted to an osmotic potential of 350 mosmol. Prior to the experiments both leaves and infiltration solutions were placed for 1 h at the same temperature as that used during the infiltration. Values are the means ± SE of four replicates. FW, Fresh weight.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

NH₄⁺ Homeostasis in Leaf Apoplastic Solution

NH₄⁺ Uptake from Leaf Apoplastic Solution

NH₄⁺ uptake in roots takes place via both a high- and a low-affinity transport system, with the former saturating at less than 1 mM (Glass et al., 1997). In the present study it was not possible to investigate NH₄⁺ uptake below 0.8 mM, because apoplastic NH₄⁺ rapidly attained a steady-state concentration of 0.8 mM, even after infiltration with an NH₄⁺-free solution (Fig. 1). Although high-affinity NH₄⁺ transport was not investigated, high levels of mRNA from the AMT1 gene, which codes for a high-affinity NH₄⁺ transporter, were found in leaves of Arabidopsis (Ninnemann et al., 1994), suggesting that a high-affinity NH₄⁺-transport system may also be present in the closely related oilseed rape species.

Neither the ATPase inhibitor DES nor the protonophore CCCP affected the net uptake of NH₄⁺ (Fig. 4a), suggesting that NH₄⁺ uptake via the low-affinity system in leaf cells of oilseed rape is independent of both plasma membrane ATPase activity and the establishment of a proton gradient. Tyerman et al. (1995) and Mouritzen and Rosendahl (1997) found that a channel-like transporter on the symbiotic interface of N₂-fixing pea transported NH₄⁺ independently of a proton gradient. In contrast, CCCP inhibited low-affinity NH₄⁺ influx in rice roots by approximately 30% (Wang et al., 1993b). The inhibition of the low-affinity NH₄⁺ uptake by K⁺ and the inhibitors La³⁺ and TEA-Cl (Fig. 3) indicates that the NH₄⁺ transport took place via a K⁺ channel or a specific NH₄⁺ channel closely related to a K⁺ channel, as was previously proposed by Ketchum and Poole (1990), Schatchtman et al. (1992), Terry et al. (1992), and Wegner et al. (1994). The fact that a 10-fold excess in apoplastic K⁺ concentration over that of NH₄⁺ inhibited NH₄⁺ net uptake by only approximately 50% (Fig. 3b) suggests a higher affinity for NH₄⁺ relative to K⁺. Since the K⁺ concentration in the leaf apoplastic solution was typically more than 10 times higher than the concentration of NH₄⁺ (data not shown), a relatively high affinity for NH₄⁺ would be required for efficient NH₄⁺ retrieval. A close relationship between an NH₄⁺ channel and a K⁺ channel would also be expected. Uozumi et al. (1995) showed that only minor site mutations in the P-domain of the inward-rectifying K⁺ channel (KAT1) from Arabidopsis expressed in yeast increased the NH₄⁺ conductance of the channel to 1 order of magnitude higher than that of K⁺.

Effect of Apoplastic pH and Temperature

The highest sensitivity of net NH₄⁺ uptake to temperature change was observed at temperatures from 10°C to 15°C (Fig. 6a), which is in agreement with results previously reported for roots of oilseed rape and rice (Macduff et al., 1987; Wang et al., 1993b). The observed increase in net NH₄⁺ uptake at increasing temperature was far too high to be caused solely by an effect on NH₄⁺ diffusion, indicating that temperature affected the transport mechanism by opening and/or closing channels (Colombo and Cerana, 1993). In accordance with the stimulating effect of temperature on NH₄⁺ net uptake, the steady-state apoplastic NH₄⁺ concentration declined (Fig. 6).

In conclusion, our data show that the apoplastic solution in leaves of oilseed rape constitutes a highly dynamic NH₄⁺ pool. NH₄⁺ is constantly added to this pool via NH₃ efflux from the mesophyll cells. The efflux of NH₃ imposes requirements for an efficient NH₄⁺-retrieval system in the leaf cell plasma membrane. This retrieval system includes a transporter with channel-like properties and is able to respond very rapidly to perturbations in apoplastic NH₄⁺ concentration, thereby maintaining apoplastic NH₄⁺ homeostasis. Documentation of the rapid NH₄⁺/NH₃ recirculation over the plasmalemma of mesophyll cells is a new contribution to understanding the dynamics and physiological implications of NH₃/NH₄⁺ transport and turnover in plants. The discovery of channel-mediated NH₄⁺ transport in leaves calls for further investigation of the genetic and molecular basis for this transport.

	FOOTNOTES

   Received April 28, 1998; accepted September 5, 1998.

	ABBREVIATIONS

Abbreviations: CCCP, carbonylcyanide-m-chlorophenylhydrazone. DES, diethylstilbestrol. TEA-Cl, tetraethylammonium chloride.

	LITERATURE  CITED

Top Abstract Introduction Methods Results Discussion References

Avery SV, Codd GA, Gadd GM (1992) Caesium transport in the cyanobacterium Anabaena variabilis: kinetics and evidence for uptake via ammonium transport systems. FEMS Microbiol Lett 95: 253-258 [CrossRef]

Colombo R, Cerana R (1993) Physiol Plant 87: 118-124 [CrossRef]

Cramer MD, Lewis OAM (1993) The influence of nitrate and ammonium nutrition on growth of wheat (Triticum aestivum L.) and maize (Zea mays L.) plants. Ann Bot 72: 359-365 [Abstract/Free Full Text]

Dentener FJ, Crutzen PJ (1994) A three dimensional model of the global ammonia cycle. J Atmos Chem 19: 331-369 [CrossRef]

Dyhr-Jensen K, Brix H (1996) Effect of pH on ammonium uptake by Typha latifolia L. Plant Cell Environ 19: 1431-1436 [CrossRef]

Farquhar GD, Firth PM, Wetselaar R, Weir B (1980) On the gaseous exchange of ammonia between leaves and the environment. Determination of the ammonia compensation point. Plant Physiol 66: 710-714 [Abstract/Free Full Text]

Finnemann J, Schjoerring JK (1998) Glutamine synthetase activity in Brassica napus L. leaves with different levels of free ammonium and amides. Plant Physiol Biochem 36: 339-346 [CrossRef]

Glass ADM, Erner Y, Kronzucker H, Schjoerring JK, Siddiqi MY, Wang M-Y (1997) Ammonium fluxes into plant roots: energetics, kinetics and regulation. J Plant Nutr Soil Sci 160: 261-268

Husted S, Schjoerring JK (1995) Apoplastic pH and ammonium concentration in leaves of Brassica napus L. Plant Physiol 109: 1453-1460 [Abstract]

Husted S, Schjoerring JK (1996) Ammonia flux between oilseed rape plants and the atmosphere in response to changes in leaf temperature, light intensity and air humidity. Interactions with stomatal conductance and apoplastic NH₄⁺ and H⁺ concentrations. Plant Physiol 112: 67-74 [Abstract]

Joy KW (1988) Ammonia, glutamine, and asparagine: a carbon-nitrogen interface. Can J Bot 66: 2103-2109

Ketchum KA, Poole RJ (1990) Pharmacology of the Ca²⁺-dependent K⁺ channel in corn protoplast. FEBS Lett 274: 115-118 [CrossRef][ISI][Medline]

Kleiner D (1981) The transport of NH₃ and NH₄⁺ across biological membranes. Biochim Biophys Acta 639: 41-52 [Medline]

Kurkdjian A, Guern J (1989) Intracellular pH: measurement and importance in cell activity. Annu Rev Plant Physiol Plant Mol Biol 40: 271-303 [CrossRef][ISI]

Langford AO, Fehsenfeld FC (1992) Natural vegetation as a source or sink for atmospheric ammonia: a case study. Science 255: 581-583 [Abstract/Free Full Text]

Leegood RC, Lea PJ, Adcock MD, Häusler RE (1995) The regulation and control of photorespiration. J Exp Bot 46: 1397-1414 [ISI]

MacDuff JH, Hopper MJ, Wild A (1987) The effect of root temperature on growth and uptake of ammonium and nitrate by Brassica napus L. cv. Bien venu in flowing solution culture. J Exp Bot 38: 53-66 [Abstract/Free Full Text]

Martin J-B, Bligny R, Rebeille F, Douce R, Leguay JJ, Mathiey Y, Guern J (1982) A ³¹P nuclear magnetic resonance study of intracellular pH of plant cells cultivated in liquid medium. Plant Physiol 70: 1156-1161 [Abstract/Free Full Text]

Mattsson M, Häusler RE, Leegood RC, Lea P, Schjoerring JK (1997) Leaf-atmosphere ammonia exchange in barley mutants with reduced activities of glutamine synthetase. Plant Physiol 114: 1307-1312 [Abstract]

Mattsson M, Schjoerring JK (1996) Ammonia emission from young barley plants: influence of N source, light/dark cycles, and inhibition of glutamine synthetase. J Exp Bot 47: 477-484

Mouritzen P, Rosendahl L (1997) Identification of a transport mechanism for NH₄⁺ in the symbiosome membrane of pea root nodules. Plant Physiol 115: 519-526 [Abstract]

Munn DA, Jackson WA (1978) Nitrate and ammonium uptake by rooted cuttings of sweet potato. Agron J 70: 312-316 [Abstract/Free Full Text]

Nakashima J, Awano T, Takabe K, Fujita M, Saiki H (1997) Immunocytochemical localization of phenylalanine ammonia-lyase and cinnamyl alcohol dehydrogenase in differentiating tracheary elements derived from Zinnia mesophyll cells. Plant Cell Physiol 38: 113-123 [Abstract/Free Full Text]

Ninnemann O, Jauniaux J-C, Frommer WB (1994) Identification of a high affinity NH₄⁺ transporter from plants. EMBO J 103: 3464-3471

Poole RJ (1978) Energy coupling for membrane transport. Annu Rev Plant Physiol 29: 437-460 [CrossRef]

Raven JA, Farquhar GD (1981) Methylammonium transport in Phaseolus vulgaris leaf slices. Plant Physiol 67: 859-863 [Abstract/Free Full Text]

Ritchie RJ (1987) The permeability of ammonia, methylamine and ethylamine in the charophyte Chara corallina (C. australis). J Exp Bot 38: 67-76 [Abstract/Free Full Text]

Schatchtman DP, Schroeder JI, Lucas WJ, Anderson JA, Gaber RF (1992) Expression of an inwardly-rectifying potassium channel by the Arabidopsis KAT1 cDNA. Science 258: 1654-1658 [Abstract/Free Full Text]

Seto-Young D, Perlin DS (1991) Effect of membrane voltage on the plasma membrane H⁺-ATPase of Saccharomyces cerevisiae. J Biol Chem 266: 1383-1389 [Abstract/Free Full Text]

Sutton M, Asman WAH, Schjoerring JK (1994) Dry deposition of reduced nitrogen. Tellus 46: 255-273 [CrossRef]

Sutton M, Schjoerring JK, Wyers P (1995) Plant-atmosphere exchange of ammonia. Philos Trans R Soc Lond-Biol Sci 351: 261-278

Terry BR, Findlay GP, Tyerman SD (1992) Direct effects of the Ca²⁺-channel blockers on plasma membrane cation channels of Amaranthus tricolor protoplast. J Exp Bot 43: 1457-1473 [Abstract/Free Full Text]

Tyerman SD, Whitehead LF, Day DA (1995) A channel-like transporter for NH₄⁺ on the symbiotic interface of N₂-fixing plants. Nature 378: 629-632 [CrossRef]

Ullrich WR, Larsson M, Larsson C-M, Lesch S, Novacky A (1984) Ammonium uptake in Lemna gibba G 1, related membrane potential changes and inhibition of anion uptake. Physiol Plant 61: 369-376 [CrossRef]

Uozumi N, Gassmann W, Cao Y, Schroeder JI (1995) Identification of strong modifications in cation selectivity in an Arabidopsis inward rectifying potassium channel by mutant selection in yeast. J Biol Chem 270: 24276-24281 [Abstract/Free Full Text]

Vessey JK, Henry LT, Chaillou S, Raper CD Jr (1990) Root-zone acidity affects relative uptake of nitrate and ammonium from mixed nitrogen sources. J Plant Nutr 13: 95-116 [Medline]

Wang MY, Glass ADM, Shaff JE, Kochian LV (1994) Ammonium uptake by rice roots. III. Electrophysiology. Plant Physiol 104: 899-906 [Abstract]

Wang MY, Siddiqi Y, Ruth TJ, Glass ADM (1993a) Ammonium uptake by rice roots. I. Fluxes and subcellular distribution of ¹³NH₄⁺. Plant Physiol 103: 1249-1258 [Abstract]

Wang MY, Siddiqi Y, Ruth TJ, Glass ADM (1993b) Ammonium uptake by rice roots. II. Kinetics of ¹³NH₄⁺ influx across the plasmalemma. Plant Physiol 103: 1259-1267 [Abstract]

Wegner LH, De Boer AH, Raschke K (1994) Properties of the K⁺ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: effect of TEA⁺, Ca²⁺, Ba²⁺ and La³⁺. J Membr Biol 142: 363-378 [ISI][Medline]

Yan F, Schubert S, Mengel K (1992) Effects of low root medium pH on net proton release, root respiration, and root growth of corn (Zea mays L.) and broad bean (Vicia faba L.). Plant Physiol 99: 415-421 [Abstract/Free Full Text]

Copyright Clearance Center:   0032-0889/98/118//08
 
© 1998 American Society of Plant Physiologists