Interactions in the uptake of amino acids, ammonium and nitrate ions in the Arctic salt-marsh grass, Puccinellia phryganodes

Authors


Correspondence: Hugh A. L. Henry. Fax: +1 416 978 5878; e-mail: hhenry@botany.utoronto.ca

ABSTRACT

The uptake of amino acids and inorganic nitrogen by roots of Puccinellia phryganodes was examined to assess the potential contribution of soluble organic nitrogen to plant nitrogen uptake in Arctic coastal marshes, where free amino acids constitute a substantial fraction of the soil-soluble N pool. Short-term excised root uptake experiments were performed using tillers grown hydroponically under controlled conditions in the field. The percentage reductions in ammonium uptake at moderate salinity (150 mm NaCl) compared with uptake at low salinity (50 mm NaCl) were double those of glycine, but glycine uptake was more adversely affected than ammonium uptake by low temperatures. Glycine uptake was higher at pH 5·7 than at pH 7·0 or 8·2. The glycine uptake was up-regulated in response to glycine, whereas ammonium uptake was up-regulated in response to ammonium starvation. Nitrate uptake was strongly down-regulated when tillers were grown on either ammonium or glycine. In contrast to N-starved roots, which absorbed ammonium ions more rapidly than glycine, the roots grown on glycine, ammonium and nitrate and not N-starved prior to uptake absorbed glycine as rapidly as ammonium and nitrate ions combined. Overall, the results indicate that amino acids are probably an important source of nitrogen for P. phryganodes in Arctic coastal marshes.

INTRODUCTION

The availability of nitrogen in forms accessible to plants is one of the primary factors that limits productivity in terrestrial ecosystems (White 1993). If plants can only utilize inorganic nitrogen uptake, as is often assumed in ecosystem-level nitrogen cycling models, then primary production is likely to be strongly affected by the rate of organic nitrogen mineralization (Giblin et al. 1991; Chapin 1995). However, recent evidence suggests that soil organic nitrogen, in the form of free amino acids, can be taken up rapidly by the roots of a wide variety of mycorrhizal and non-mycorrhizal plants, ranging from Arctic and alpine grasses, sedges and shrubs (Chapin, Moilanen & Kielland 1993; Kielland 1994; Raab, Lipson & Monson 1996, 1999), to boreal grasses, trees and shrubs (Näsholm et al. 1998), temperate crop plants (Schobert & Komor 1987, Jones & Darrah 1994; Näsholm, Huss-Danell & Högberg 2001) and tropical trees and shrubs (Turnbull et al. 1996). Direct uptake of amino acids by plant roots may uncouple primary production from nitrogen mineralization rates in ecosystems in which free amino acids are readily available relative to soluble inorganic nitrogen (Chapin 1995). Hence, it calls into question the validity of nitrogen cycling models for these systems (Chapin 1995).

Although free amino acid concentrations are often orders of magnitude lower than inorganic nitrogen concentrations in temperate soils, they account for a relatively large proportion of total available nitrogen for plant growth in Arctic systems, in which low temperatures, low precipitation and a short growing season result in slow mineralization rates and a build-up of soluble organic nitrogen (Nadelhoffer et al. 1992; Kielland 1995; Atkin 1996). In Arctic coastal marshes, inorganic nitrogen availability is further limited by high salinity due to the inhibitory effects of chloride ions on nitrification (McClung & Frankenberger 1985; Wilson et al. 1999). High salinity also has a strong inhibitory effect on inorganic nitrogen uptake, particularly the uptake of nitrate (Cram 1973; Deane-Drummond & Glass 1982), although ammonium uptake is inhibited as well (Bradley & Morris 1990; Chambers, Mozdzer & Ambrose 1998). Rates of N uptake also diminish at low temperatures, although ammonium absorption is less sensitive to suboptimal growth temperatures than nitrate absorption in some species (Clarkson, Hopper & Jones 1986; Clarkson, Jones & Purves 1992). Amino acids may be an important supplementary source of nitrogen in Arctic coastal marshes, provided that the inhibitory effects of sodium chloride and low temperatures on amino acid uptake are not severe.

Soluble nitrogen concentrations in Arctic coastal marsh soils are highly variable, both in space and time (Henry & Jefferies 2002). This variability in nitrogen supply may have important implications for the relative rates of amino acid uptake in these systems, because the concentration of nitrogen substrates in soil solutions strongly influences their individual rates of uptake by roots. Root transport systems of inorganic nitrogen are regulated to respond rapidly to fluctuations in inorganic nitrogen sources (Glass et al. 1999). For example, high affinity ammonium transport is up-regulated in response to N-starvation and down-regulated in the presence of ammonium ions (Lee & Rudge 1986; Kronzucker, Siddiqi & Glass 1996). There is also a need for plants to integrate signals from several potential nitrogen pools in order to regulate total nitrogen uptake (Glass et al. 1999). This can lead to the favouring of one form of nitrogen over another, as in ammonium-dominated systems, in which the presence of ammonium ions inhibits nitrate transport in some species (Kronzucker, Siddiqi & Glass 1997). Despite widespread evidence for the direct uptake of amino acids by roots, the role of external nitrogen sources in the regulation of amino acid transport systems is unknown, yet essential for estimating relative rates of amino acid uptake where different N sources are available.

The majority of ecological studies of amino acid uptake by plants are based on glycine uptake, as glycine is typically present at relatively high concentrations in the soil solution and can be taken up rapidly due, in part, to its low molecular weight relative to other amino acids (Kielland 1994; Lipson et al. 1999). However, in Arctic coastal marsh soils, alanine, proline and glutamic acid concentrations are higher than those of glycine and several other amino acids are present at concentrations equal to those of glycine (Henry & Jefferies 2002). Although a range of specific and general amino acid transporters are expressed in plant roots (Fischer et al. 1998), it is not known to what extent multiple transporters may alleviate competition between amino acids for transport sites, and how rapidly mixtures of amino acids are taken up relative to glycine alone.

In the present study, factors affecting the relative rates of amino acid and inorganic nitrogen uptake in the Arctic salt-marsh grass, Puccinellia phryganodes, were examined. Excised roots of tillers grown hydroponically under controlled conditions in the field were used to compare the uptake kinetics of 15N-labelled amino acids, ammonium and nitrate ions and to quantify the effects of salinity, low temperatures, variation in pH and competition between nitrogen sources on uptake rates. Excised root experiments were also conducted to examine the regulation of amino acid and inorganic nitrogen uptake when tillers were grown on different nitrogen substrates. The combined goal of these experiments was to assess the capability of the salt-marsh grass to utilize amino acids as a supplementary source of nitrogen under conditions similar to those in coastal soils.

METHODS

Study species and site description

Puccinellia phryganodes (Trin.) Scribn. and Merr. is a stoloniferous, halophytic graminoid that dominates intertidal salt-marshes along the Hudson Bay coast and is common in salt marshes in circumpolar regions (Hultén 1968). Net above-ground productivity of intact swards in coastal marshes of the Hudson Bay lowland is estimated to range between 100 and 150 g m−2 year−1 (Cargill & Jefferies 1984b; Hik & Jefferies 1990). Increases in primary productivity following addition of inorganic nitrogen to swards indicate that the availability of this element limits plant growth (Cargill & Jefferies 1984a). Soils in the intertidal zone have a greyish mineral horizon of marine sediment in which the upper 2·5 cm contains organic material. Over the growing season, from late May to late July, the mean amino acid concentrations in soil solutions in the intertidal zone range from 32 to 45 µm, and ammonium and nitrate concentrations range from 55 to 160 and 10 to 31 µm, respectively; however, soluble N concentrations obtained from individual soil samples can be as much as an order of magnitude higher (Henry & Jefferies 2002). Amino acids that are present at high concentrations are alanine, proline, glutamic acid, leucine, tyrosine, gamma-butyric acid and glycine. Soil pH averages 7·12 ± 0·04 (SE, n= 24), and the salinity of extracted soil solutions can rise to as high as 1·5 m NaCl in late summer in intertidal sites devoid or nearly devoid of vegetation, although NaCl concentrations typically range from 50 to 250 mm under high biomass swards of vegetation (Srivastava & Jefferies 1995; Wilson & Jefferies 1996).

Excised root experiments

Tillers of Puccinellia phryganodes were collected from a salt-marsh sward at La Pérouse Bay, Wapusk National Park, 25 km east of Churchill, Manitoba (58·43°N, 94·28°W). All tillers were collected from the same 1 m × 1 m patch in order to minimize genetic variability. Each shoot was approximately 2·5 cm high and root length was 3–5 cm. Each growing season new roots replace those produced during the previous growing season. The tillers were stripped of old root material and placed in an aerated solution containing one-tenth strength Hoagland's macronutrients (ammonium nitrate as the N source) (Hoagland & Arnon 1950), 0·2 mm Fe-EDTA and 50 mm NaCl at a density of 12 tillers per 4 L container. The nutrients were dissolved in calcium/magnesium-rich river water due to the limited availability of deionized water in the field. The river water was fast-flowing, well-aerated, free of suspended organic matter and contained no detectable ammonium or nitrate (water was sampled weekly; the lower detection limit of N was 0·7 µm). Containers (black high-density polyethylene) were placed in a herbivore exclosure in a supratidal marsh at La Pérouse Bay and sunk into the soil to maintain water temperature within 1 °C of soil temperature at rooting depth. The tillers were grown hydroponically for 5–6 weeks and solutions were replaced every four days. Based on the rate of N accumulation in plant tissue, the draw-down of N from growth solutions was < 5% over 4 d. The pH of growth solutions remained over this period at approximately 7·5. Forty-eight hours prior to the start of uptake experiments, the tillers were starved of nitrogen by immersing the roots in 0·5 mm CaCl2 and 50 mm NaCl dissolved in river water.

The availability of root material for experiments was limited given the slow growth rate of our study species (the dry weight of roots of each tiller obtained from the field was < 2 mg). Root excision allowed us to pool roots of a large number of tillers and apportion precise quantities of root to each replicate. Roots were excised, blotted and weighed into samples of 180 mg. Based on a modified method of Epstein, Schmid & Rains (1963), samples were placed in cheese cloth ‘teabags’, kept in an aerated holding solution of 0·5 mm CaCl2 and 50 mm NaCl and allowed to equilibrate at the temperature of the experimental solution for 20 min (deionized water was used throughout all steps of the uptake experiments). After equilibration, roots were placed for 20 min in 200 mL of a well-aerated solution identical to the holding solution but containing a 15N-labelled substrate (Sigma-Aldrich, St Louis, MO, USA; specific activity of 99%). Pilot trials using ammonium and glycine indicated that the relationship between total uptake and time was linear for at least the first 50 min of uptake after excision. The pH of the incubation solutions was 7·0. Following absorption, the roots were rinsed for 5 min in a solution of 5 mm KCl to remove label left in the Donnan free space, then dried at 60 °C. Dried root material was ground to a fine powder and analysed for its N content and 15N/14N ratio relative to unenriched control samples using an Isochrom continuous flow stable isotope mass spectrometer (Micromass, Manchester, UK) coupled to an elemental analyzer (CHNS-O EA1108; Carlo Erba, Rodano, Italy). Isotopic analyses were performed by the University of Waterloo Environmental Isotope Laboratory. Data of the 13C/12C ratio and C content of tissues (see below) were also obtained.

In order to characterize the uptake kinetics, uptake of 15N-labelled glycine, aspartic acid, glutamic acid, leucine, 15NH4Cl and K15NO3 was measured for two replicates at concentrations of 0, 10, 12, 15, 20, 30, 55 and 500 µm at 18 °C. This range of concentrations was selected to give a uniform coverage of points on a reciprocal axis. Uptake of a mixture of equal concentrations of 15N-labelled common salt-marsh amino acids (glycine, aspartic acid, glutamic acid, leucine, proline and alanine) was also measured, and uptake of 13C15N-labelled glycine was examined to determine whether the amino acid was taken up intact into roots. For both the amino acid mixture and 13C15N-labelled glycine, total amino acid concentration ranged from 0 to 500 µm (as above).

The 15N-glycine and 15NH4Cl uptake rates at a higher salinity (150 mm NaCl) were compared with those at the same range of external 15N concentrations used for uptake trials at 50 mm NaCl (n = 2). The 15N-glycine and 15NH4Cl uptake rates at external 15N concentrations of 10 and 100 µm (n = 3) were also measured at 2, 5 and 18 °C (n = 3) and at a pH of 5·7, 7·0 and 8·2, where the external 15N concentration was 100 µm (n = 3). Sodium phosphate monobasic and HCl were titrated to obtain pH 5·7, sodium phosphate dibasic and NaOH were titrated to obtain pH 8·2 and both forms of sodium phosphate were titrated to obtain pH 7·0. All pH treatments received equal concentrations of sodium phosphate and roots were pH acclimated for 2 weeks prior to excision (additions of Na and Cl from NaOH and HCl were less than 2% of their concentrations in the background solutions). Substrate competition trials were conducted by measuring uptake of 15N-labelled substrates in the presence of 100 µm of unlabelled NH4Cl, NaNO3, glycine, aspartic acid, glutamic acid or a mixture of amino acids (glycine, glutamic acid, aspartic acid, leucine, alanine, proline and lysine mixed equally to give a total final concentration of 100 µm). A total concentration of 100 µm was selected for competing substrates as it approximates to concentrations typically present in soil solutions under field conditions. The uptake of 15N-labelled substrates was measured for two replicates at concentrations ranging from 0 to 500 µm (see above).

Methods used to examine the regulation of N uptake by the presence of N substrates departed slightly from methods used for other uptake trials. Tillers were grown on 50 mm NaCl and one-tenth strength nitrogen-free Hoagland's solution to which was added one of the four following nitrogen substrates: glycine, NH4Cl, NaNO3 or an equal mixture of all three substrates. Tillers were assigned at random to the different treatments. The total N concentration of each growth solution was 1·6 mm, equal to that of one-tenth strength Hoagland's solution and solutions were changed every two days. After 6 weeks of growth, tillers grown on a given nitrogen source were randomly re-assigned to growth on either glycine, or NH4Cl or NaNO3 (all 1·6 mm N). Uptake of the newly introduced nitrogen source was measured at a concentration of 100 µm15N for roots excised after 0, 1, 3 and 7 d (n = 3). On day 0, tillers grown on an equal mixture of glycine, NH4Cl, and NaNO3 were used as a control. On days 1, 3 and 7, excised roots from tillers grown on the same substrate as the labelled N source, but in the absence of other N substrates, were used as controls. Roots were not starved prior to excision for substrate regulation trials.

Data analyses

For excised root experiments, maximum uptake rate (Vmax) and the half saturation constant (Km) were estimated by fitting ordinary least squares regression lines to double reciprocal plots of uptake rate versus concentration. Regression lines were back-transformed to a linear scale for curve fitting. For salinity trials (Table 1) and substrate competition between glycine, NH4Cl and NaNO3 (see Table 4), predicted uptake rates at a concentration of 100 µm15N, a typical N concentration present in salt-marsh soils, were interpolated from regression lines for the purpose of comparison. In the amino acid competition trials (see Table 5), the difference between uptake of an amino acid at an external concentration of 100 µm15N in the absence and presence of 100 µm N mixed cocktail of amino acids was expressed as a percentage of the difference between uptake of the amino acid alone at 100 µm15N and one half of its uptake at 200 µm15N. This value, termed ‘percentage competition’ in the context of this study, expresses uptake of an amino acid in terms of the outcome of competition from other amino acids at comparable concentrations of total soluble N.

Table 1.  Uptake kinetics of 15N-glycine and 15N-ammonium by excised roots of Puccinellia phryganodes at low and moderate salinity (50 and 150 mm NaCl, respectively)
Substrate[NaCl]Km
(µmol L−1)
Vmax
(µmol g−1 h−1)
r2Uptake rate at 100 mm 15N
(µmol g−1 h−1)
  1. Affinity (Km), maximum uptake rate (Vmax) and the correlation coefficient (r2) were estimated by plotting uptake rate against labelled substrate concentration in a double reciprocal (Lineweaver–Burk) plot. Uptake rate at 100 µm15N was interpolated from regression for the purposes of comparison.

15N-glycine5063·022·30·9813·7
15N-glycine15056·517·10·9810·9
15N-ammonium5036·175·20·9955·3
15N-ammonium15029·146·31·0035·9
Table 4.  Uptake kinetics of 15N-glycine and 15N-ammonium ions by excised roots of Puccinellia phryganodes in the presence and absence of unlabelled competing nitrogen sources
SubstrateCompeting substrate
(100 µm)
Km
(µmol L−1)
Vmax
(µmol g−1 h−1)
r2Uptake rate at 100 µm15N
(µmol g−1 h−1)
  1. All tillers were grown on NH4NO3. Km, Vmax, r2 and uptake rate at 100 µm were estimated as in Table 1.

15N-glycine46·015·20·9710·4
15N-glycineAmmonium60·914·80·91 9·2
15N-glycineNitrate38·514·30·9610·3
15N-ammonium63·170·40·9143·2
15N-ammoniumGlycine26·644·80·7535·4
15N-ammoniumNitrate20·950·80·7542·0
Table 5.  Uptake kinetics of 15N-glycine, 15N-glutamic acid and 15N-aspartic acid by excised roots of Puccinellia phryganodes in the presence and absence of a mixture of 100 µm unlabelled amino acids (a.a) (equal concentrations of alanine, aspartic acid, glutamic acid, glycine, leucine, lysine and proline)
SubstrateCompeting ion
(100 µm)
Km
(µmol L−1)
Vmax
(µmol g−1 h−1)
r2Percentage competition
at 100 µm15N
Ki
(µm)
  1. Km, Vmax and r2 were estimated as in Table 1. Percentage competition at 100 mm15N was calculated by expressing the difference between uptake of the amino acid in the absence and presence of 100 µm mixed amino acids as a percentage of the difference between uptake at 100 µm and one half of uptake at 200 µm in the absence of competing amino acids. Dissociation constants (Ki) are also displayed.

15N-glycine23·715·30·91n.a.n.a.
15N-glycinea.a. mixture50·212·90·93 69·011·7
15N-glutamic acid14·2 8·10·68n.a.n.a.
15N-glutamic acida.a. mixture33·8 7·10·90 54·0 7·5
15N-aspartic acid22·1 7·50·67n.a.n.a.
15N-aspartic acida.a. mixture29·1 4·20·70105·0 6·6

RESULTS

Uptake of 15N by excised roots

The uptake kinetics of ammonium, nitrate and amino acids by roots N-starved for 48 h prior to uptake approached saturation at low external concentrations (10–55 µm) (Fig. 1a). With the exception of ammonium uptake, in which the standard error was relatively large, curves obtained from double reciprocal plots underestimated uptake at 500 µm. The most rapid rates of amino acid uptake were exhibited by glycine, which was taken up from 20 to 50% faster than leucine, glutamic acid and aspartic acid at concentrations between 10 and 55 µm, the range of concentrations typically encountered in salt-marsh soils (Fig. 1b). When roots were incubated in an equi-mixture of 15N-labelled alanine, aspartic acid, leucine, glutamic acid, glycine and proline, 15N was taken up at a rate comparable to that of 15N-glycine alone over the same range of concentrations (Fig. 1b). The uptake of 13C from 13C15N-labelled glycine indicated that this amino acid was taken up intact; however, rates of 13C uptake were only one-third to one-half those of 15N uptake (Fig. 1a). Ammonium ions were taken up three to four times as fast as glycine over the range of concentrations tested, whereas the uptake rates of nitrate were low, and the process saturated at very low concentrations relative to the uptake of amino acids (Fig. 1a). Based on rates of 15N uptake, draw-down of 15N in incubation solutions was < 5% (with the exception of low concentration ammonium solutions, which were drawn down by as much as 15% by N-starved roots).

Figure 1.

Plots of uptake rate versus substrate concentration for excised roots of P. phryganodes provided with 15N and 13C-labelled substrates in short-term uptake experiments (all tillers grown on NH4NO3 and N-starved 48 h prior to uptake). Curves were obtained by back-transforming OLS linear fits to double reciprocal plots of uptake rate versus concentration. For each substrate, the following estimates of Km (µm) and Vmax (µmol g−1 h−1) were obtained, respectively: 15N-glycine (37·1, 13·5), 15N-ammonium (57·6, 66·5), 15N-nitrate (1·4, 6·2), 13C-glycine (56·6, 7·5), 15N-glutamic acid (66·2, 13·2), 15N-aspartic acid (42·2, 8·7), 15N-leucine (21·7, 8·9), mixture of 15N-alanine, aspartic acid, glutamic acid, glycine, leucine and proline (13·8, 9·9). Error bars denote standard error.

Effects of salinity, temperature, pH and competition between N sources on uptake rates

At an external N concentration of 100 µm, the rate of glycine uptake was 20% lower at moderate salinity (150 mm) than at low salinity (50 mm NaCl), whereas the rate of ammonium uptake was 35% lower at moderate salinity than at low salinity (Table 1). In contrast, the difference between uptake at 2 or 5 °C relative to uptake at 18 °C was greater for glycine than for ammonium (Table 2). Ammonium uptake rates increased with increasing alkalinity of the external solution, whereas glycine uptake rates increased with increasing acidity (Table 3). In competition trials between glycine, ammonium and nitrate, the presence of 100 µm nitrate had no detectable effect on rates of glycine uptake, and glycine uptake at an external 15N concentration of 100 µm was reduced by 12% in the presence of 100 µm ammonium ions (Table 4). Likewise, the interference of nitrate with rates of ammonium uptake was minimal, but decreases in ammonium uptake of 18% occurred in the presence of glycine (Table 4). Estimates of percentage competition between a mixture of unlabelled, common salt-marsh amino acids (total concentration of 100 µm) and individual amino acids, 15N-glycine, 15N-glutamic acid and 15N-aspartic acid, each at an external 15N concentration of 100 µm, were 69, 54 and 105%, respectively (Table 5).

Table 2.  Uptake rates of 10 and 100 µm15N-glycine and 15N-ammonium ions by excised roots of Puccinellia phryganodes at 2, 5 and 18 °C
Temperature (°C)
10 µm
Mean (SE)
100 µm
Mean (SE)
10 µm
Mean (SE)
100 µm
Mean (SE)
  1. Means and standard errors (SE) were calculated for each treatment (n = 3).

21·00 (0·09)2·83 (0·03)13·78 (1·55)40·08 (4·52)
51·63 (0·08)5·50 (0·24)21·04 (0·79)59·56 (3·14)
183·54 (0·13)8·93 (0·66)27·26 (2·18)69·75 (8·30)
Table 3. . Uptake rates of 100 µm15N-glycine, 15N-ammonium and 15N-nitrate by excised roots of Puccinellia phryganodes at a pH 5·7, 7·0 and 8·2
pH15N-glycine
Mean (SE)
15N-ammonium
Mean (SE)
15N-nitrate
Mean (SE)
  1. Means and standard errors (SE) were estimated for each treatment (n = 3).

 5·718·3 (0·3)61·8 (6·5) 9·9 (2·5)
 7·013·2 (0·8)63·8 (11·2)10·2 (1·7)
10 4 (0·7)76·1 (9·4) 8·6 (1·1)

Regulation of nitrogen uptake

Uptake of glycine by tillers grown on an equal mixture with respect to N of glycine, NH4Cl and NaNO3 exceeded that of tillers grown solely on ammonium or nitrate at day 0 (Fig. 2a). For tillers grown on ammonium and nitrate, rates of glycine uptake increased from day 0 to day 3, but subsequently decreased to intermediate values from day 3 to day 7. Differences in glycine uptake between control (glycine-grown) tillers at days, 1, 3 and 7 were not significant [one-way analysis of variance (anova), d.f. = 2, 6, F = 2·26, P = 0·19]. Tillers grown in the absence of ammonium, particularly those grown solely on nitrate, took up ammonium rapidly on day 0 relative to plants grown on a mixture of all three substrates (Fig. 2b). Rates of ammonium uptake by nitrate- and glycine-grown tillers rapidly decreased by day 1. Differences in ammonium uptake between control (ammonium-grown) tillers at days 1, 3 and 7 were not significant (one-way anova, d.f. = 2, 6, F = 2·91, P = 0·13). For nitrate uptake, uptake rates at day 0 were very low for tillers grown solely on ammonium or glycine or on a mixture of all three substrates (Fig. 2c). On day 1, uptake of nitrate by glycine- and ammonium-grown tillers remained low relative to uptake of nitrate by tillers grown solely on nitrate. Nitrate uptake increased for glycine- and ammonium-grown tillers between day 3 and day 7. Differences in nitrate uptake between control (nitrate-grown) tillers at days 1, 3 and 7 were not significant (one-way anova, d.f. = 2, 6, F = 0·90, P = 0·45).

Figure 2.

Figure 2.

Plots of (a) 15N-glycine uptake; (b) 15N-ammonium uptake; and (c) 15N-nitrate uptake by excised roots of Puccinellia phryganodes at an external 15N concentration of 100 µm (note that Y-axis scales differ among plots). Treatments (see symbol key in figure) refer to the nitrogen source tillers were grown on until day 0. The mixed treatment refers to tillers grown on equal concentrations of glycine, ammonium and nitrate at the same total nitrogen concentration as other nitrogen treatments. Thereafter, tillers were grown on the same nitrogen source as used in 15N uptake trials. Tillers were not N starved prior to uptake. In each panel, control tillers (i.e. initial N source was the same as the 15N source) are denoted by bold lines interconnecting points. Points indicate mean and standard error (n = 3).

Figure 2.

Figure 2.

Plots of (a) 15N-glycine uptake; (b) 15N-ammonium uptake; and (c) 15N-nitrate uptake by excised roots of Puccinellia phryganodes at an external 15N concentration of 100 µm (note that Y-axis scales differ among plots). Treatments (see symbol key in figure) refer to the nitrogen source tillers were grown on until day 0. The mixed treatment refers to tillers grown on equal concentrations of glycine, ammonium and nitrate at the same total nitrogen concentration as other nitrogen treatments. Thereafter, tillers were grown on the same nitrogen source as used in 15N uptake trials. Tillers were not N starved prior to uptake. In each panel, control tillers (i.e. initial N source was the same as the 15N source) are denoted by bold lines interconnecting points. Points indicate mean and standard error (n = 3).

DISCUSSION

Rates of amino acid uptake by excised roots of P. phryganodes were within the range of those reported in the literature for Arctic and alpine plants (Chapin et al. 1993; Kielland 1994; Raab et al. 1996, 1999). Uptake kinetics of amino acids were characterized by a high-affinity component that saturated at low external amino acid concentrations (Fig. 1a & b). Based on the tendency for fitted curves to underestimate the uptake of the different nitrogen sources at 500 µm, it is probable that a non-saturable, low-affinity component contributes to uptake at higher concentrations, as first described by Epstein, Rains & Elzam (1963) for K+ transport. This combination of low- and high-affinity transport systems is analogous to root transport systems typically present for ammonium and nitrate (Glass et al. 1999) and is supported by the cloning of both low- and high-affinity root amino acid transporters in Arabidopsis (see Fischer et al. 1998). Although root excision may reduce rates of N uptake relative to intact plants, such reductions are not commonly observed within 1–2 h following excision (Bloom & Caldwell 1988; Huang et al. 1992). For Puccinellia, uptake remained linear up to at least 50 min following excision, which indicates that excision-induced reductions in uptake did not occur over the time course of the incubations. Roots may also exhibit a pulse in N efflux following excision; however, this effect is transient and net uptake recovers within several minutes (Aslam et al. 1996).

Across the range of concentrations tested, the rate of glycine uptake by N-starved roots was approximately one-third that of ammonium ions and nitrate uptake was minimal (Fig. 1a). This result contrasts with that obtained for non-starved roots grown on an equal mixture of all three N substrates, in which glycine uptake equalled the combined uptake of ammonium and nitrate (Fig. 2– see below). The uptake of 13C when roots were provided with 13C15N-glycine indicated that at least a portion of each amino acid was probably taken up intact and not mineralized at the root surface. The 13C uptake was only up to 50% as high as 15N uptake, which indicates that some amino acids may have been de-aminated prior to uptake of the 15N moiety. Puccinellia phryganodes is a sterile tripliod and must be cultured from tillers as opposed to seed. Therefore, although the uptake media were sterile, traces of bacterial contamination may have been present on the surface of roots. However, examination of roots under a light microscope showed no accumulation of slime on roots and no evidence of the build-up of bacterial plaque on the root surface under high magnification. In many algae and fungi, extracellular de-amination of amino acids can occur at the cell surface, followed by uptake of ammonium ions (Paul & Cooksey 1979, 1981; DeBusk et al. 1981; Munoz-Blanco, Hidalgo-Martínez & Cárdenas 1990). Although extracellular deaminases were not localized on the surface of root cell membranes, hypothetically, their presence could explain the low 13C incorporation of 13C15N-amino acids from hydroponic media. The 13C may be underestimated in 13C15N-amino acid uptake experiments because of the high background quantity of 13C in root tissue relative to 15N and the large error associated with this dilution of the 13C signal (Näsholm & Persson 2001; Nordin, Högberg & Näsholm 2001). Losses of 13C may also occur as a result of root respiration (Schimel & Chapin 1996), but substantial respiratory losses of 13C would have been unlikely in the present study given the short incubation time.

Despite the capability of roots to take up a wide range of amino acids, it has been suggested that glycine is the only amino acid taken up rapidly relative to inorganic nitrogen due to its neutral charge and low molecular weight (Kielland 1994; Lipson et al. 1999). If correct, this would limit the contribution of amino acids to plant nitrogen nutrition in salt-marsh systems, in which total amino acid concentrations are comparable with ammonium concentrations but comprised of only 3–4% glycine N, on average (Henry & Jefferies 2002). In the present study, the uptake rate of a mixture of 15N-amino acids was comparable with that of glycine alone between 10 and 55 µm and uptake rates of leucine, glutamic acid and aspartic acid (each tested separately) were at least half of those of glycine over the same range of concentrations (Fig. 1a & b). Therefore, other amino acids besides glycine probably contribute substantially to total amino acid uptake, particularly when their combined concentration greatly exceeds that of glycine. In substrate competition trials, the uptake of individual 15N-amino acids did decrease somewhat in the presence of a mixture of unlabelled amino acids at typical field concentrations, suggesting that the uptake rates of individual amino acids tested in isolation are not simply additive (Table 5). However, when the total amino acid concentration was held constant, the effect of competition from other amino acids on the uptake of an amino acid was less severe, on average, than the relative decline in the rate of uptake associated with its saturation kinetics at higher concentrations (200 µm). These results provide indirect evidence for the expression of multiple root amino acid transporters with some degree of specificity, as described in Fischer et al. (1998).

Uptake trials at moderate salinity (150 mm NaCl) revealed that percentage reductions in ammonium uptake rates relative to uptake at low salinity (50 mm NaCl) were double those of glycine (Table 1). In contrast, reductions in uptake rates at low temperatures were higher for glycine than for ammonium ions (Table 2). These results suggest that the relative contribution of amino acid uptake to total nitrogen uptake may increase as the growing season progresses due to the presence of higher salinities and warmer temperatures (soil temperatures reach 12–14 °C at a depth of 5 cm and can exceed 20 °C close to the soil surface). These rapid rates of uptake would be enhanced by the relatively high concentrations of amino acids present in the soil solution at this time (Henry & Jefferies 2002). Biomass production and nitrogen acquisition are also highest from mid- to late growing season due to warm temperatures, further increasing the demand for amino acids as a nitrogen source in P. phryganodes.

The rapid uptake of glycine by roots of P. phryganodes at pH 5·7 is consistent with the pH optima for the uptake of amino acids by barley roots, which range, for the most part, from pH 4 to pH 6 (Soldal & Nissen 1978). In contrast to glycine, ammonium uptake was relatively low at pH 5·7 relative to uptake at neutral or alkaline pH. This trend has also been shown for Lolium perenne, where the uptake rate of glycine relative to that of ammonium was greater at pH 6 than at pH 9 (Thornton 2001). The pH of salt-marsh soil solution is neutral and temporal and spatial variability in pH are low (Wilson & Jefferies 1996). However, plants that take up nitrogen primarily as ammonium (as opposed to nitrate) tend to lower the pH of the rhizosphere, such that it is more acidic than the pH of the bulk soil solution (Nye 1981). Therefore, it is conceivable that root acidification may favour glycine uptake over ammonium uptake in P. phryganodes.

The presence of 100 µm ammonium in excised root solutions over a 20 min time course decreased glycine uptake by only 12%, and ammonium uptake was reduced by a comparably low amount in the presence of glycine (Table 4). Therefore, it does not appear that competition between inorganic and organic nitrogen sources would substantially alter their relative rates of uptake. However, results from the nitrogen substrate regulation trials revealed that uptake of nitrogen by roots of P. phryganodes is highly sensitive to the presence of amino acids, ammonium and nitrate in the external growth solution over the time scale of 1 to 3 d. Uptake of glycine by roots grown on a mixture of glycine, ammonium and nitrate exceeded uptake of glycine by roots grown solely on ammonium or nitrate previously (Fig. 2a), which provides evidence that glycine uptake is up-regulated in the presence of glycine. This evidence was further supported by the increase in glycine uptake by plants initially grown on ammonium or nitrate after three days of exposure to glycine. Rates of glycine uptake by ammonium and nitrate grown plants at day zero were comparable to those rates of glycine uptake exhibited by ammonium or nitrate grown tillers starved 48 h prior to root excision (Fig. 1a). Hence, it does not appear that glycine uptake is up-regulated by nitrogen starvation.

In contrast to glycine uptake, ammonium uptake was highly up-regulated in the absence of ammonium, particularly for roots grown solely on nitrate, and subsequently down-regulated after one day of exposure to ammonium (Fig. 2b). Rapid up-regulation of ammonium in response to nitrogen starvation and down-regulation in response to the presence of ammonium or amino acids in the external solution has been demonstrated in many species (Lee & Rudge 1986; Morgan & Jackson 1988; Kronzucker et al. 1996; Glass et al. 1999). This property of ammonium regulation has important implications for the estimation of relative rates of amino acid and ammonium uptake under field conditions. Uptake data derived from ammonium nitrate-grown roots starved for 48 h prior to excision indicate that ammonium uptake is from three to four times greater than glycine uptake for any given concentration (Fig. 1a). Nevertheless, these data are an overestimation of ammonium uptake, which is rapidly up-regulated in response to nitrogen starvation and (they may also underestimate glycine uptake, which appears to be down-regulated in the absence of glycine). In contrast, when tillers were supplied with all three nitrogen sources simultaneously and not starved prior to excision, glycine uptake slightly exceeded ammonium uptake (Fig. 2). However, these tillers were grown at nitrogen concentrations almost an order of magnitude higher than those typically encountered in salt-marsh soils, which could have resulted in the down-regulation of ammonium uptake. Although the relative uptake rates of glycine and ammonium presumably lie between these two extremes, the estimation of nitrogen concentrations experienced by roots in the field is not a trivial exercise. Estimations of nitrogen concentrations in the soil solution often are overestimates of nitrogen concentrations at the root surface, due to the formation of diffusion gradients close to the root surface (Nye 1969), and the effect of rhizosphere microbial activity (Schobert, Kockenberger & Komor 1988; Lipson & Monson 1998; Jones 1999). Furthermore, roots likely experience pulses of high soluble N concentrations as a result of rapid microbial and root cell lysis following freeze-thaw cycles (Hobbie & Chapin 1996), or as a result of goose faecal deposition (Ruess, Hik & Jefferies 1989). Although mean soil solution concentrations of N in free amino acids, ammonium and nitrate ions combined range from 100 to 200 µm over the growing season (n = 18), mean concentrations early in the season and concentrations of individual soil samples throughout the growing season can be an order of magnitude higher (Henry & Jefferies 2002).

In the absence of ammonium or glycine, nitrate uptake was up-regulated by the presence of nitrate in the external solution (Fig. 2), consistent with the induction pattern of high affinty nitrate transport exhibited by most species (Lee 1982; Siddiqi et al. 1990; Kronzucker, Siddiqi & Glass 1995). However, nitrate uptake was minimal in the presence of glycine or ammonium ions. The down-regulation of nitrate uptake in the presence of ammonium has been demonstrated for other species in ammonium-dominated systems (Kronzucker et al. 1997). It appears from the results of excised root experiments that the combined presence of ammonium, amino acids and high salinities in salt-marsh soils is likely to restrict nitrate uptake and result in a very minor contribution of nitrate to plant nutrition. Amounts of this N source are low in these saline soils in any event (Wilson & Jefferies 1996).

Overall, the results of this study provide evidence that amino acids are an important supplementary source of nitrogen for the growth of P. phryganodes. Roots of P. phryganodes respond rapidly to fluctuations in external nitrogen concentrations and edaphic conditions, and the directions and strengths of these responses on rates of nitrogen uptake differ between the nitrogen sources (Fig. 3). Based on the results of excised root experiments, amino acid uptake is likely at its highest relative to ammonium uptake from the mid- to the late period of the growing season when soil salinity, soil temperatures and free amino acid concentrations in the soil solution are high. A thorough understanding of the dynamics of both soluble organic and inorganic nitrogen is essential for estimating plant nitrogen availability in Arctic coastal marshes and elsewhere.

Figure 3.

Summary of effects high salinity, low temperature, acidic pH and external concentrations of glycine, ammonium and nitrate on rates of uptake of glycine, ammonium and nitrate in Puccinellia phryganodes. Plus symbols denote up-regulation and minus symbols denote down-regulation. Smaller ovals indicate relatively low effect. The lack of an arrow indicates that no effect was present, with the exception of the effect of the relative effect of low temperature on nitrate uptake, which was not tested. A strong negative down-regulation of nitrate uptake under high salinity was assumed.

ACKNOWLEDGMENTS

This work was supported by the Natural Sciences and Engineering Research Council of Canada through a research grant to R.L.J. and a postgraduate scholarship to H.A.L.H. Additional funding was provided by the Association of Canadian Universities for Northern Studies and the Northern Scientific Training Program. We thank Dawn Davidson, Rachel Sturge, Michael Warnock and researchers at the La Pérouse Bay Field Station and staff at Wapusk National Park for assistance and logistical support. We are grateful for the comments of two anonymous reviewers and the Associate Editor that led to an improvement in the quality of the manuscript.

Received 16 May 2002; received in revised form 28 August 2002; accepted for publication 3 September 2002

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