Interactions between uptake of amino acids and inorganic nitrogen in wheat plants

Abstract. Soil-borne amino acids may constitute a source of nitrogen (N) for plants in various terrestrial ecosystems but their importance for total N nutrition is unclear, particularly in nutrient-rich arable soils. One reason for this uncertainty is lack of information on how the absorption of amino acids by plant roots is affected by the simultaneous presence of inorganic N forms. The objective of the present study was to study absorption of glycine (Gly) and glutamine (Gln) by wheat roots and their interactions with nitrate (NO3−) and ammonium (NH4+) during uptake. The underlying hypothesis was that amino acids, when present in nutrient solution together with inorganic N, may lead to down-regulation of the inorganic N uptake, thereby resulting in similar total N uptake rates. Amino acids were enriched with double-labelled 15N and 13C, while NO3− and NH4+ acquisition was determined by their rate of removal from the nutrient solution surrounding the roots. The uptake rates of NO3− and NH4+ did not differ from each other and were generally about twice as high as the uptake rate of organic N when the different N forms were supplied separately in concentrations of 2 mM. Nevertheless, replacement of 50% of the inorganic N with organic N was able to restore the N uptake to the same level as that in the presence of only inorganic N. Co-provision of NO3− did not affect glycine uptake, while the presence of glycine down-regulated NO3− uptake. The ratio between 13C and 15N were lower in shoots than in roots and also lower than the theoretical values, reflecting higher C losses via respiratory processes compared to N losses. It is concluded that organic N can constitute a significant N-source for wheat plants and that there is an interaction between the uptake of inorganic and organic N.


Introduction
Amino acids are ubiquitously present in the soil solution and may constitute a significant source of N for plants in terrestrial ecosystems.The concentration of amino acids in the soil solution ranges from 0.1 to 60 µM and typically constitutes 10-40 % of the total soluble N (Hill et al., 2011;Jamtgard et al., 2008;Jones et al., 2002).The pool of dissolved amino acids is supplemented by a substantial pool of absorbed amino acids which can be up to 50 times higher than the amount of free amino acids and, in unfertilized soils, even higher than the content of ammonium and nitrate (Jamtgard et al., 2010).In cropping systems which rely on recycling and decomposition of organic N sources (e.g.animal manure based, low-input or organic agriculture), amino acids may represent a large N input (El-Naggar et al., 2009) and an important plant-available N pool (Jones et al., 2002).Under such circumstances, a significant proportion of total N in the soil solution consists of dissolved organic N, including both free amino acids and complex, humic-rich soluble organic matter which has relatively low bioavailability (Christou et al., 2005).The distribution of free amino acids in soil may show considerable spatial heterogeneity due to, e.g.degradation of sloughed-off root cell in the rhizosphere, leading to hotspots with greatly higher concentrations than in the bulk soil (Jones et al., 2002).The fast turnover of amino acids in soils implies that soil solution pool can be continuously renewed and, hence, provide the basis for significant plant uptake despite a low concentration in soil solution (Thornton and Robinson, 2005).
One aspect causing uncertainty about the importance of organic N compounds as N source for plants is the possible interaction between organic and inorganic N forms during absorption by plant roots (Thornton and Robinson, 2005).The existing knowledge about these interactions is mainly based on experimental approaches in which plants have been pretreated with amino acids and subsequently exposed to NO − 3 or NH + 4 or vice-versa (e.g.Persson and Nasholm, 2002).In most cases, inorganic N absorption is down-regulated following pre-treatment with amino acids, although the actual responses are quite variable and may also depend on the N form used during the pre-treatment (Thornton, 2004).Less information is available from experiments in which inorganic and organic N forms have been supplied together.Using 14 C/ 13 C/ 15 N triple-labelling instead of the double 15 N-13 C or 15 N-14 C labelling classically used for direct root uptake experiments, Rasmussen and Kuzyakov (2009) and Rasmussen et al. (2010) recently showed that the simultaneous uptake of inorganic C derived from mineralization of organic compounds interfered with root uptake of dual-labelled organic N. It will be of similar importance to assess the interactions between organic and inorganic forms during absorption by plant roots in order to establish the significance of organic N compounds for plant N nutrition.
The purpose of the present work was to evaluate interactions between the amino acids glycine and glutamine and the inorganic N forms nitrate and ammonium during uptake by plant roots.The underlying hypothesis was that amino acids, when co-present in nutrient solution with inorganic nitrogen, may lead to a down-regulation of inorganic N uptake or viceversa, thereby resulting in similar total N uptake rates.A series of experiments was conducted in which double labelled ( 15 N-13 C) amino acids applied to wheat plants were used to trace amino acid absorption, while the net-uptake rate of inorganic N was determined by regular sampling and analysis of the nutrient solution.

Materials and methods
Three independent experiments were carried out as detailed below.The purpose of Experiment I was to test the hypothesis that partial substitution of an inorganic N source (NO − 3 or NH + 4 ) with an organic form (glycine or glutamine) would be able to restore the total N uptake to the same level as that in the presence of only inorganic N. In Experiment II the objective was to test how organic N (glycine) affected inorganic N (NO − 3 ) uptake, and vice-versa, when the concentration of each of the two sources was the same whether supplied alone or in mixture (i.e.increasing total N concentration when the two sources were applied together).Finally, the purpose of Experiment III was to test the hypothesis that plants were able to sustain growth and N acquisition when inorganic N (NO − 3 ) was completely replaced by an organic N source (glycine, glutamine, arginine or asparagine).

Experiment I
Spring wheat (Triticum aestivum L. cv.Amaretto) was germinated in vermiculite for 10 days at 27 O, and 10 mM EDTA.The plants were grown in a greenhouse with controlled environment at 250 to 280 µmol m −2 s −1 photon flux density and a 18 • C/16 • C day/night (16/8 h) temperature regime.The nutrient solution was replaced every week and the position of the cultivation units shifted to minimize differences in growth due to micro-environmental differences.
After 30 days in the basic nutrient solution, plants from 4 cultivation units (16 individual plants) were harvested while the remaining 36 units were allocated to nine different nitrogen treatments.The nitrogen treatments lasted for 60 h and consisted of (i) 2 mM glycine (Gly), (ii) 2 mM glutamine (Gln), (iii) 1 mM Gly + 1 mM NO − 3 , (iv) 1 mM Gln + 1 mM NO − 3 , (v) 1 mM Gly + 1 mM NH (ix) no nitrogen (N 0 ) with four replicates.Amino acids were enriched by 1.37 atom % with double-labelled 15 N and 13 C (Icon Services Inc., New York, USA).Both the amide and amine N atom in Gln were 15 N labelled.Ammonium was supplied as (NH 4 ) 2 SO 4 and nitrate as KNO 3 .The other plant nutrients were provided at the same concentrations as in the basic nutrient solution except for Ca 2+ and SO 2− 4 which were used to obtain cation-anion balance.The pH in the nutrient solution was maintained around 5.5 by use of calcium hydroxide or sulphuric acid every 12 h.Twenty ml samples of the nutrient solution were taken from each cultivation unit at 4 time points, viz.0, 22, 45 and 60 h after beginning of the treatment period.Plants were harvested by the end of the 60 h experimental period.

Experiment II
Wheat plants were pre-grown for 30 days under the same conditions as above.Plants were then subjected to addition of 1 mM glycine and 3 mM NO − 3 or 1 mM glycine alone (4 replicates).Prior to the N treatments, 4 groups with each 4 plants had not received N for 3 days (N starvation treatment).The glycine was 10 % enriched with double-labelled 15 N and 13 C. N uptake from glycine and NO − 3 was then measured over 3 days.

Experiment III
Under the same light and temperature conditions as Experiments I and II, wheat plants were grown for 7 days in complete nutrient solutions with NO − 3 as the sole N source followed by a 17 day period in which 1 mM unlabelled glycine, glutamine, arginine, asparagine or 3 mM NO − 3 were provided as the only N source (4 replicates).

Analysis of plants and nutrient solution
At harvest, the plant material in each cultivation unit were quickly separated into roots and shoots which were weighed, frozen in liquid N and stored at −80 • C. Prior to analysis all plant material was freeze-dried until constant weight and milled to <0.5 mm.The dried samples were subsequently used for analyzing total C, 13 C/ 12 C, total N and 15 N/ 14 N by mass spectrometry in a system consisting of an ANCA-SL elemental analyzer coupled to a 20-20 tracermass mass spectrometer (SerCon Ltd., Crewe, UK).
All samples of nutrient solutions were analysed for NO − 3 and NH + 4 using flow injection analysis (Lachat 8000 series, Hach, Loveland, Colorado). 15NH + 4 , 15 NO − 3 and total N were in selected cases measured by mass spectrometry following the micro-diffusion technique (Brooks et al., 1989).In order to double check the recovery of amino acids in solution, CF-IRMS analyses of the nutrient solutions sampled at the completion of the experiments were performed after oven-drying at 50 • C.

Calculations
N recoveries and N uptake rates were calculated on the basis of excess 15 N and 13 C in the harvested plant material combined with the concentrations of N left in the nutrient solution.
N and C atom % excess was calculated as excess compared to the mean atom % of the pre-treatment plants, according to the equation: where APE = atom percent excess, AP p = atom percent in treated plant, and AP ptp = atom percent in pre-treatment plants (average).
The amount of amino-acid derived 15 N or 13 C taken up by the plant was calculated by use of Eq. ( 2): where AA-15 N = amino acid-derived 15 N in plant tissue (mg), DW = plant dry weight (mg), N% = plant nitrogen concentration (mg mg −1 ), and APE(N) = nitrogen atom percentage excess (mg mg −1 ).Recovery of the added amino acids in plant material was calculated by use of Eq. ( 3): where R N = N recovery in plant (%) and AA-15 N add = added amino acid-15 N (mg).
For inorganic N, the uptake rate was calculated based on the N left in solution according to Eq. ( 4), and for organic N based on the 15 N excess in plant material after harvest, according to Eq. ( 5): where UR = uptake rate and T 2 , T 1 = sampling times (h after start of treatments).All calculations of NO − 3 and NH + 4 uptake rates were based on the initial linear part of the depletion curves extending up to 45 h after start of the experiment.

Statistical analysis
Statistical analysis was carried out using R version 2.12.2 (www.r-project.org)through mixed linear model comparisons and analysis of variance followed by Tukey's and Duncan's test.The model comparisons were based on pair-wise analysis of variance between two statistical models.Whenever two compared models were statistically different (95 % confidence interval), the model with higher complexity was accepted.

N content and 13 C: 15 N ratio
15 N and 13 C excess increased in plants supplied with amino acids (Table 1).The excess of the two stable isotopes was almost twice as high in plants receiving organic N (2 mM Gly or Gln) as the sole N supply compared to plants receiving only half the concentration of organic N (1 mM Gly or Gln) in combination with inorganic N (1 mM NH + 4 or NO − 3 ).The N treatments did not affect the dry matter weight of the plants which on average was 2.7 ± 0.1 g plant −1 (P > 0.05; data not shown).Similarly, the N concentration in the plant dry matter and the total N content did not differ (P > 0.05) among Nfed plants irrespective of in which form the N was supplied (Table 1).
The ratio between 13 C and 15 N recovered in the plant tissues was lower than the theoretical values of 2.5:1 for glutamine and 2:1 for glycine in all plants receiving organic N (Fig. 1).The 13 C: 15 N ratio was in all cases lower in shoots than in roots (P ≤ 0.05).Plants receiving glutamine as the sole N supply had higher 13 C: 15 N ratio in the roots than plants supplied with glycine, reflecting the higher C:N ratio of glutamine.However, the opposite was the case for shoots, showing higher 13 C: 15 N ratio for plants receiving glycine relative to glutamine (Fig. 1).Irrespective of the amino acid supplied, shoot 13 C: 15 N ratios were higher in plants receiving NH + 4 compared to NO − 3 (P ≤ 0.05).

N uptake rates
The uptake rates of NO − 3 and NH + 4 did not differ from each other and were generally about twice as high as the uptake rate of organic N when the different N forms were supplied separately in concentrations of 2 mM, with the exception of glycine and NH + 4 (Fig. 2).Nevertheless, replacement of 50 % of the inorganic N with organic N was able to restore the N uptake to the same level as that in the presence of only inorganic N (Fig. 2).
Significantly different amino acid uptake rates were obtained depending on whether 13 C or 15 N were used for the calculations (Fig. 3).In all cases, 13 C-based uptake rates were much lower than those based on 15 N.

Interactions between N forms
The model-comparison statistical analysis of the data in Fig. 2 showed a significant interaction between the uptake of organic and inorganic N (P ≤ 0.05).Such interaction was also evident in Experiment II, where co-provision of NO − 3 did not affect glycine uptake, while the presence of glycine down-regulated NO − 3 uptake (Fig. 4).Pre-starvation of plants did not lead to increased uptake of glycine (Fig. 4).
Plants exposed to glycine as the sole N form after precultivation on nitrate had lower NO − 3 concentrations in roots as well as shoots compared to plants continuously receiving NO − 3 (Fig. 5).Pre-starvation also led to a reduction in tissue NO − 3 (Fig. 5).Tissue NH + 4 concentrations were similar across the different N treatments (Fig. 5).

N and C recovery in wheat plants
About 70-80 % of the added amino-acid-N was recovered in the plants.In all cases, at plant harvest there was still a small fraction of N left in the solutions containing amino acids (Fig. 6).Labelled 15 NH + 4 was detected in some of the originally pure amino acid solutions, indicating possible deamination by microorganisms in solution or efflux of 15 NH + 4 from the roots.However, deamination prior to uptake did not seem prominent as evidenced by the fact that when plants in Experiment III were grown for an extended period with amino acids as the sole N source, plant growth was reduced by 50 % or more compared to plants grown in NO − 3 (Fig. 7).

Interaction between organic and inorganic N forms
Inorganic nitrogen is considered to be the preferred N form taken up by higher plants.This was also the case in the present study, as shown by generally higher uptake rates of NO − 3 and NH + 4 compared to amino acids when the total N concentration was the same (i.e. 2 mM; Fig. 2).Compared across experiments, the uptake rate of glycine in wheat roots seemed only to respond marginally to an increase in external concentration, being similar at 1 mM (Fig. 4) and 2 mM (Fig. 2) when glycine was supplied as the sole N source.In contrast, the uptake of NO − 3 was higher at 3 mM (around 28 µmol g −1 root DW h −1 ; Fig. 4) compared Table 1.N concentration and total N content of plants in Experiment I. Numbers are mean values (n = 4).Letters indicate significant differences at P = 0.05 (Tukey's test).Within columns, values followed by the same letter are not significantly different.Excess 15 N % and 13 C ‰ values were calculated taking the N 0 treatment as reference ( 15 N % = 0.3706 for roots and 0.3681 for shoots; 13 C % = 1.0786 for roots and 0.0773 for shoots).to 2 mM (around 23 µmol g −1 root DW h −1 ; Fig. 2).Assuming that NO − 3 uptake responds linearly to changes in the external NO − 3 concentration in the low-affinity range (Glass, 2009) this would imply a net uptake rate of around 18 µmol g −1 root DW h −1 at 1 mM external NO − 3 .The actual observed uptake rate of NO − 3 in solutions containing 1 mM NO − 3 in the presence of 1 mM glycine was only around 13 µmol g −1 root DW h −1 (Fig. 2), indicating that  NO − 3 uptake was down-regulated.This conclusion based on data from Experiment I is corroborated by the data obtained in Experiment II, showing that co-provision of 1 mM glycine and 3 mM NO − 3 resulted in a down-regulation of NO − 3 uptake while that of glycine was unaffected (Fig. 4).Since glycine uptake is not down-regulated in the presence of NO − 3 , plants may be able to maintain a similar total N uptake as when NO − 3 constitutes their sole N source, as shown by the fact that replacement of 50 % of the inorganic N with organic N in Experiment I was able to restore the N uptake rate to the same level as that in the presence of only inorganic N (Fig. 2).Plants may thus be able to fulfil their N requirement when organic N is provided as an N source in mixture with NO − 3 .Ammonium has been reported to cause down-regulation of amino acid uptake and vice-versa (Henry and Jefferies, 2003;  15 N, 13 C double-labelled glycine (1 mM) was applied during a 3-day period with or without 3 mM NO − 3 to hydroponically grown wheat plants pre-cultivated on NO − 3 .Data are means ± SE (n = 4) obtained in Experiment II and letters indicate significant differences at P = 0.05.Thornton and Robinson, 2005) although in some cases this may not appear (Rodgers and Barneix, 1993).

N excess
The interaction between root uptake of inorganic and organic N forms reflects a feed-back repression in which the amino acid glutamine represents an important signal for the shoot to communicate N status to the roots, thereby enabling plants to regulate their rate of N uptake to accommodate for N demand during plant growth (Girin et al., 2007;Nazoa et al., 2003).Nitrate starvation may relieve this feed-back repression (Krapp et al., 1998) but did not lead to increased uptake of glycine or nitrate in the present work (Fig. 4).The feed-back repression is primarily targeted at transport proteins involved in NO − 3 and NH + 4 uptake, explaining why glycine uptake may not be affected to the same extent.Despite being taken up at a high rate, amino acids cannot constitute the sole N supply without leading to growth depression (Fig. 7).This may reflect bottlenecks in the rate at which nitrogen is transferred to other essential amino acids via deamination and or transamination.In addition, signalling pathways controlling phytohormone balance may be impeded causing growth inhibition (Krouk et al., 2011).More information is needed on growth responses of plants to mixtures of amino acids in concentrations typically present in soil solution.

Fate of absorbed C and N in the plant
The ratio between excess 13 C and 15 N in roots and shoots was significantly lower than the theoretical values of 2.5 and 2 in glutamine and glycine (Fig. 1).It has been argued that if the enrichment of 13   relationship upon exposure to double-labelled amino acids, then the amino acids have been absorbed as intact molecules (Jones et al., 2005).However, some of the absorbed amino acid C will always be lost in the form of CO 2 produced during deamination and breakdown of the C skeleton in the TCA cycle (Nasholm and Persson, 2001) or in processes related to photorespiration (Bauwe et al., 2010).In wheat plants this may amount to 60-80 % of the absorbed amino acid C (Hill et al., 2011), agreeing with the losses reported in our study (Fig. 6).Substantial amounts of 15 N can be lost from plant shoots (Schjoerring et al., 1989).These losses are typically highest towards plant maturation and may partly be due to N volatilization (Schjoerring et al., 2002;Sommer et al., 2004).However, the gaseous N losses from the foliage generally amount to only 1-5 % of the N present in the plant, i.e. being considerably lower than C losses due to respiration.A lower 13 C: 15 N excess ratio does therefore not necessarily imply that amino acids were not taken up in intact form as also concluded by Schimel and Chapin (1996).Lower excess 13 C: 15 N ratios in plant tissues relative to applied amino acids might also result from deamination of amino acids before uptake, followed by uptake of inorganic 15 N.It does not seem likely that this process contributed significantly in our experiments, because only a few percentages of the 13 C were recovered in solution at the end of the experiment (Fig. 6).If the amino acids had been decomposed by microorganisms and the N released in inorganic form to the solution and subsequently taken up by the roots, a significant portion of the labelled C would have remained in the bacteria without being lost by respiration during the 3 day experimental period.The utilization efficiency of easily decomposable substrates typically varies between 0.4-0.7 within the first days of decomposition (Parton et al., 1987;Steinweg et al., 2008;Thiet et al., 2006).This means that the amount of C lost by microbial respiration would be roughly similar to the amount retained in the microbial biomass.Consequently, taking into account the fact that only a few percentage of 13 C was recovered in the solution at the end of the 60-h experimental period (Fig. 6), losses of C following microbial decomposition are only likely to have been responsible for a few percentages of the unrecovered 13 C.Our assumption that the majority of the 13 C loss occurred after uptake of intact amino acids is further corroborated by the fact that the growth of plants exposed to amino acids as the sole N source was more than 50 % reduced (Fig. 7) which would not have been the case if a substantial deamination in the nutrient solution had made NH + 4 available as N source.Finally, amino acids are chemically very stable in water and would not deaminate spontaneously under the experimental conditions we applied.
The transport of amino acids from roots to shoots in plants is dominated by the amides glutamine and asparagine (Finnemann and Schjoerring, 1999;Harrison et al., 2000).Before being utilized as a N-source, a substantial part of the glycine absorbed by roots is therefore probably deaminated and channeled into amide synthesis, a process accompanied by release and re-assimilation of NH + 4 .Glutamine may on the other hand be transported directly to shoots and utilized directly as an N source in the glutamate synthase-glutamine synthetase cycle.Alternatively, a glutamine transaminase may transfer the amino nitrogen to glycine or other amino acids (Joy, 1988).These amino acids may act as substrates for aminotransferases transferring the amino group to glyoxalate, thereby forming glycine which enters the photorespiratory pathway, resulting in release of CO 2 and NH + 4 (Bauwe et al., 2010).The fact that the excess 13 C: 15 N ratio was much lower in shoots compared to roots (Fig. 2) suggests that photorespiration may have played an important role in the assimilation of the N derived from the absorbed amino acids.Despite the release of NH + 4 in the various metabolic processes involved in processing the absorbed amino acids, plants fed with glycine or glutamine did not contain elevated tissue NH + 4 levels in neither roots nor shoots (Fig. 5).The capacity for re-assimilation of the generated NH + 4 was thus not exceeded.

Methodological aspects
Two different methods, viz.stable isotope labelling with 15 N-13 C amino acids and NO − 3 /NH + 4 depletion in the nutrient solution, were used to measure the separate contribution of organic and inorganic N sources to total plant N acquisition.Technically, the two methods differ in that with the first plants are harvested destructively at the end of the experimental period and subsequently analysed to obtain the uptake of the isotopically labelled compound, while with the second the nutrient solution in the root medium is sampled and analysed at different time intervals to determine the rate of solute removal by the roots.However, the two methods basically reflect the same underlying process viz.root uptake, and they will give similar results, as proven by Persson and Nasholm (2002), provided that other experimental and analytical factors are not interfering.Uptake rates of isotopically labelled amino acids might be underestimated if N losses occur during their assimilation in the plant, while on the other hand inorganic N uptake rate could be overestimated if there were N losses from the solution via e.g.volatilization.As discussed in Sect.4.2 we do not consider these sources of error to have had a major impact on our results.Rasmussen and Kuzyakov (2009) and Rasmussen et al. (2010) used 14 C/ 13 C/ 15 N triple-labelling to show that the simultaneous uptake of inorganic C interfered with root uptake of dual-labelled organic N. Unfortunately, a similar methodological option is not available for N because the only isotope that can be implemented is 15 N.The radio-isotope 13 N with a half-life just below 10 min has been used for measurement of short-term uni-directional fluxes in plant roots (Ter Steege et al., 1998) but cannot be used in studies that also aim at revealing interactions associated with N assimilation and growth responses, which are manifested over a period of time longer than a few minutes or hours.There is a methodological challenge in proving the uptake of intact amino acids in experiments where their uptake is studied over an extended period of time.Persson and Nasholm (2002) used different metabolic inhibitors to prevent assimilation of the absorbed amino acids in order to analyse their accumulation inside the plant.However, this approach may lead to secondary feedback processes affecting the absorption of both inorganic and organic N forms.

Conclusions
It is concluded that amino acids can constitute a significant N source for wheat plants and that there is an interaction between the uptake of inorganic and organic N. Glycine uptake is not down-regulated in the presence of NO − 3 , while NO − 3 uptake is reduced in the presence of glycine.Nitrogen prestarvation appears to counteract the down regulation of NO − 3 uptake in the presence of glycine.The 13 C: 15 N excess ratio is lower in shoots than in roots and is also lower than in the provided source of double-labelled amino acid, reflecting higher C losses via respiratory processes compared to N losses.The observed interactions between root absorption of inorganic and organic N forms have implications for further assessment of the contribution of organic N compounds to plant N nutrition.

Figure 1 . 575 Fig. 1 .
Figure 1.Ratio between 13 C: 15 N excess in roots and shoots of wheat plants supplied with double-labelled amino acids, either alone or mixed with inorganic N. Values are means (n=4) obtained in Experiment I and letters indicate significant differences at P = 0.05 (Duncan test).

Figure 2 .
Figure 2. Mean N uptake rates in wheat plants supplied with different combinations of amino acids and inorganic N forms in Experiment I.For inorganic-N, values were calculated on the basis of the N remaining in solution, while for amino acid-N values 580 were calculated based on 15 N content in plants.All treatments had a total N concentration of 2 mM.Error bars indicate cumulative standard error (n=4), and letters indicate significant differences at P = 0.05 (Tukey's test).

Fig. 2 .
Fig. 2. Mean N uptake rates in wheat plants supplied with different combinations of amino acids and inorganic N forms in Experiment I.For inorganic-N, values were calculated on the basis of the N remaining in solution, while for amino acid-N values were calculated based on 15 N content in plants.All treatments had a total N concentration of 2 mM.Data are means, error bars indicate cumulative ± SE (n = 4), and letters indicate significant differences at P = 0.05 (Tukey's test).

Figure 3 .
Figure 3. Amino acid uptake rates calculated on the basis of either 15 N or 13 C data in Experiment I. Error bars indicate standard error (n=4).

Fig. 3 .
Fig.3.Amino acid uptake rates calculated on the basis of either 15 N or 13 C data in Experiment I. Data are means ± SE (n = 4) and letters indicate significant differences at P = 0.05.

Figure 4 .Fig. 4 .
Figure 4. Glycine (A) and nitrate uptake (B) in wheat plants. 15N, 13 C double-labelled glycine (1 mM) was applied during a 3-day period with or without 3 mM NO 3 -to hydroponically grown wheat plants pre-cultivated on NO 3 -.Data are means ± SE (n=4) obtained in Experiment II and letters indicate significant differences at P = 0.05.

Figure 6 .
Figure 6.Recoveries in plant roots, plant shoots and solution based on plant and solution analyses in Experiment I: (A) 15 N; (B) 13 C. Error bars indicate total standard 630 error (n=4) and letters significant differences at P = 0.05.

Fig. 6 .
Fig. 6.Recoveries in plant roots, plant shoots and solution based on plant and solution analyses in Experiment I: (A) 15 N; (B) 13 C. Data are means ± SE, error bars indicate cumulative SE (n = 4) and letters significant differences at P = 0.05.

Figure 7 .Fig. 7 .
Figure 7. Growth (A) and N concentration (B) of wheat plants supplied different amino acids as sole N source during a 17-day period in Experiment III.Data are mean 655 values ± SE (n=4) and letters indicate significant differences at P = 0.05.