Elevated carbon dioxide increases nitrate uptake and nitrate reductase activity when tobacco is growing on nitrate, but increases ammonium uptake and inhibits nitrate reductase activity when tobacco is growing on ammonium nitrate

Authors

  • P. Matt,

    1. Botanisches Institut, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany and
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    • *Present address: Max Planck Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany.

  • M. Geiger,

    1. Botanisches Institut, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany and
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  • P. Walch-Liu,

    1. Institut für Pflanzenernährung, Universität Hohenheim, 70593 Stuttgart, Germany
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  • C. Engels,

    1. Institut für Pflanzenernährung, Universität Hohenheim, 70593 Stuttgart, Germany
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  • A. Krapp,

    1. Botanisches Institut, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany and
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    • Present address: Institute Nationale de la Recherche Agronomique, Centre de Versailles, Route de St Cyr, 78026 Versailles Cedex, France.

  • M. Stitt

    1. Botanisches Institut, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany and
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    • *Present address: Max Planck Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany.


Correspondence: Mark Stitt. Fax: + 49 331 567 8101; e-mail: mstitt@mpimp-golm.mpg.de

Abstract

The influence of elevated [CO2] on the uptake and assimilation of nitrate and ammonium was investigated by growing tobacco plants in hydroponic culture with 2 mm nitrate or 1 mm ammonium nitrate and ambient or 800 p.p.m. [CO2]. Leaves and roots were harvested at several times during the diurnal cycle to investigate the levels of the transcripts for a high-affinity nitrate transporter (NRT2), nitrate reductase (NIA), cytosolic and plastidic glutamine synthetase (GLN1, GLN2), the activity of NIA and glutamine synthetase, the rate of 15N-nitrate and 15N-ammonium uptake, and the levels of nitrate, ammonium, amino acids, 2-oxoglutarate and carbohydrates. (i) In source leaves of plants growing on 2 mm nitrate in ambient [CO2], NIA transcript is high at the end of the night and NIA activity increases three-fold after illumination. The rate of nitrate reduction during the first part of the light period is two-fold higher than the rate of nitrate uptake and exceeds the rate of ammonium metabolism in the glutamate: oxoglutarate aminotransferase (GOGAT) pathway, resulting in a rapid decrease of nitrate and the accumulation of ammonium, glutamine and the photorespiratory intermediates glycine and serine. This imbalance is reversed later in the diurnal cycle. The level of the NIA transcript falls dramatically after illumination, and NIA activity and the rate of nitrate reduction decline during the second part of the light period and are low at night. NRT2 transcript increases during the day and remains high for the first part of the night and nitrate uptake remains high in the second part of the light period and decreases by only 30% at night. The nitrate absorbed at night is used to replenish the leaf nitrate pool. GLN2 transcript and glutamine synthetase activity rise to a maximum at the end of the day and decline only gradually after darkening, and ammonium and amino acids decrease during the night. (ii) In plants growing on ammonium nitrate, about 30% of the nitrogen is derived from ammonium. More ammonium accumulates in leaves during the day, and glutamine synthetase activity and glutamine levels remain high through the night. There is a corresponding 30% inhibition of nitrate uptake, a decrease of the absolute nitrate level, and a 15–30% decrease of NIA activity in the leaves and roots. The diurnal changes of leaf nitrate and the absolute level and diurnal changes of the NIA transcript are, however, similar to those in nitrate-grown plants. (iii) Plants growing on nitrate adjust to elevated [CO2] by a coordinate change in the diurnal regulation of NRT2 and NIA, which allows maximum rates of nitrate uptake and maximum NIA activity to be maintained for a larger part of the 24 h diurnal cycle. In contrast, tobacco growing on ammonium nitrate adjusts by selectively increasing the rate of ammonium uptake, and decreasing the expression of NRT2 and NIA and the rate of nitrate assimilation. In both conditions, the overall rate of inorganic nitrogen utilization is increased in elevated [CO2] due to higher rates of uptake and assimilation at the end of the day and during the night, and amino acids are maintained at levels that are comparable to or even higher than in ambient [CO2]. (iv) Comparison of the diurnal changes of transcripts, enzyme activities and metabolite pools across the four growth conditions reveals that these complex diurnal changes are due to transcriptional and post-transcriptional mechanisms, which act several steps and are triggered by various signals depending on the condition and organ. The results indicate that nitrate and ammonium uptake and root NIA activity may be regulated by the sugar supply, that ammonium uptake and assimilation inhibit nitrate uptake and root NIA activity, that the balance between the influx and utilization of nitrate plays a key role in the diurnal changes of the NIA transcript in leaves, that changes of glutamine do not play a key role in transcriptional regulation of NIA in leaves but instead inhibit NIA activity via uncharacterized post-transcriptional or post-translational mechanisms, and that high ammonium acts via uncharacterized post-transcriptional or post-translational mechanisms to stabilize glutamine synthetase activity during the night.

Abbreviations:
GLN1

cytosolic glutamine synthetase

GLN2

plastidic glutamine synthetase

NIA

nitrate reductase

NRT2

high-affinity nitrate transporter

Introduction

The response to elevated [CO2] depends on the nitrogen supply (Stitt & Krapp 1999). When nitrogen is low or marginal, the acceleration of growth in elevated [CO2] drives the plant into a nitrogen deficiency (Coleman, McConnaughay & Bazzaz 1993; Nie et al. 1995; Rogers et al. 1996a, b; Geiger et al. 1999). This is revealed by lower levels of nitrate, decreased nitrate reductase (NIA) activity, lower levels of amino acids (especially glutamine) and a lower protein content (Geiger et al. 1999; Makino & Mae 1999; Stitt & Krapp 1999). There is a gradual decline or ‘acclimation’ of photosynthesis (Coleman et al. 1993; Nie et al. 1995; Makino & Mae 1999; Stitt & Krapp 1999; Geiger et al. 1999), and the stimulation of growth is attenuated or abolished (Tissue, Thomas & Strain 1993; Johnson, Ball & Walker 1995; Bowler & Press 1996; Rogers et al. 1996a, b; Geiger et al. 1999). When nitrogen is high, elevated [CO2] leads to a sustained stimulation of photosynthesis (Wong 1979; Pettersson,macDonald & Stadenburg 1993; Sage 1994; Geiger et al. 1999) and stimulation of growth (Ferrario-Mery et al. 1997; Geiger et al. 1998, 1999). Nitrate, amino acids and protein remain high, revealing that the uptake and utilization of inorganic nitrogen has been increased.

Increased sugar levels lead to higher levels of the transcripts for nitrate and ammonium transporters (Gojon et al. 1998; Gazzarrini et al. 1999; von Wirén et al. 2000a, b). Although this provides a potential mechanism to stimulate nitrogen uptake in elevated [CO2], empirical studies reveal a more complex picture. Although elevated [CO2] increased nitrate uptake per unit root weight in loblolly pines (Bassirirad et al. 1996) and Prosopis glandulosa (Bassirirad et al. 1997), it did not alter nitrate uptake in Nardus agrostis (Bassirirad et al. 1997) and it inhibited nitrate uptake in a mixed field community (Jackson & Reynolds 1996). Ammonium uptake was unaltered in elevated [CO2] in loblolly pine (Bassirirad et al. 1996) and decreased in a mixed field community (Jackson & Reynolds 1996). The response may depend on the nitrogen supply: for example, elevated [CO2] inhibited nitrate uptake in loblolly pine growing on low nitrate, whereas uptake was stimulated when the plants were growing on high nitrate (Larigauderie, Reynolds & Strain 1994). Elevated [CO2] has complex effects on mineral nitrogen availability, due to altered rates of utilization (see above) and altered mobility in the soil (Conroy & Hocking 1993, Van Vuuren et al. 1997).

The higher levels of sugars and lower levels of glutamine in elevated [CO2] (see above) should promote transcription and post-translational activation of NIA and promote nitrate assimilation (Stitt & Krapp 1999). However, this will be counteracted if nitrate is depleted (Geiger et al. 1999; Stitt & Krapp 1999). This may explain why NIA activity sometimes increases slightly (Maevskaya et al. 1990; Sharma & Sen Gupta 1990) and sometimes decreases (Hocking & Meyer 1991a, b; Purvis, Peters & Hageman 1974; Ferrario-Mery et al. 1997) in elevated [CO2]. Transfer of Plantago major to elevated [CO2] resulted in a transient increase in NIA activity that was reversed during the next 4 d (Fonseca, Bowsher & Stulen 1997). In tobacco, elevated [CO2] led to a decrease of NIA activity when the nitrate supply was barely adequate for maximal growth at ambient [CO2], and stimulated nitrate assimilation when nitrate was supplied in excess (Geiger et al. 1999). The stimulation was due to modified diurnal regulation, leading to higher activity later in the light period and at night (Geiger et al. 1998).

Even less is known about ammonium metabolism in elevated [CO2]. This is surprising because this is one of the sites where elevated [CO2] could have a direct beneficial influence on metabolism. Reassimilation of photorespired ammonium accounts for 90% of the flux through the glutamate: oxoglutarate aminotransferase (GOGAT) pathway in leaves of C3 plants in ambient [CO2], and the lower rate of photorespiration in elevated [CO2] should release capacity for de novo nitrogen assimilation. This presupposes that the expression and activity of the enzymes in the GOGAT pathway remain high in elevated [CO2]. Although 800 p.p.m. [CO2] led to a small decrease of the transcripts for cytosolic (GLN1) and plastid (GLN2) glutamine synthetase in tomato leaves (von Wiren et al. 2000a), 2000 p.p.m. [CO2] had no effect on expression of GLN2 or glutamate:oxoglutarate aminotransferase in tobacco leaves (Migge et al. 1997). The accompanying changes in enzyme activity, and the effect of elevated [CO2] in roots, where most de novo ammonium assimilation occurs, have not been investigated. Geiger et al. (1999) found that elevated [CO2] leads to a decrease of NIA activity in tobacco growing on surplus ammonium nitrate, in contrast to nitrate where (see above) NIA activity remains high. They proposed that elevated [CO2] preferentially stimulates ammonium uptake and/or assimilation, leading to accumulation of reduced nitrogen and repression of NIA.

Nitrate uptake and nitrate and ammonium assimilation are closely interlinked processes. None of the previous investigations provides an integrated picture of their response to elevated [CO2]. A recent study (Matt et al. 2001), in which nitrate uptake, nitrate assimilation and ammonium metabolism were investigated in tobacco plants growing in hydroponic culture with super-optimal (2 mm) nitrate in ambient [CO2], provides the starting point for the following paper. In these standard conditions, the rate of nitrate assimilation in leaves during the first part of the light period exceeds the rate of nitrate uptake by a factor of two, and also exceeds the rate of amino acid synthesis and export. As a result, nitrate is depleted and the immediate products of nitrate assimilation accumulate, including ammonium, glutamine and the photorespiratory intermediates glycine and serine. This imbalance is reversed later in the day and during the night, when nitrate assimilation decrease and nitrate uptake and glutamine synthetase activity remain high or increase.

In parallel with the plant material described in Matt et al. (2001), tobacco plants were grown in 1 mm ammonium nitrate and ambient [CO2], 2 mm nitrate and elevated [CO2], and 1 mm ammonium nitrate and elevated [CO2]. The first aim was to investigate the long-term effect of ammonium on the nitrate uptake and assimilation in ambient [CO2]. Although it is well established that ammonium leads to a rapid repression of nitrate transport and NIA (Hoff, Truong & Caboche 1994; Krapp et al. 1998; Forde & Clarkson 1999), little is known about the long-term consequences for nitrogen metabolism. The second aim was to investigate the influence of elevated [CO2] on nitrogen metabolism in plants growing with nitrate as the sole nitrogen source. We have already reported that the diurnal regulation of NIA activity is altered in elevated [CO2] (Geiger et al. 1998), but nothing is known about the accompanying changes of nitrate uptake and ammonium metabolism. The third aim was to investigate whether the response to elevated [CO2] is modified when ammonium is present in the growth medium. In particular, we wanted to investigate whether elevated [CO2] leads to a preferential stimulation of ammonium uptake and utilization, and whether this leads to an inhibition of nitrate uptake and metabolism. Finally, we anticipated that this large data matrix would provide information about regulation mechanisms.

Materials and methods

Plant cultivation

Tobacco (Nicotiana tabacum cv. Gatersleben) was grown in a climate chamber under controlled environmental conditions with a 12 h light period (light intensity of 500–600 µE m−2 s−1), a 25 °C/20 °C light/dark temperature regime and 60% relative humidity. From the beginning of the experiment, the plants were cultivated under two different atmospheric CO2 concentrations, ambient (400 p.p.m.) and elevated (800 p.p.m.). Seeds were germinated in a mixture consisting of 90% (w/w) peat culture substrate (Euflor GmbH, München, Germany), 7% (w/w) perlite and 3% (w/w) sand. Fourteen days after sowing, plants were transferred to an aerated hydroponic culture system in a 5 L pot, and supplied with saturated CaSO4 solution during the first 24 h, and thereafter with a full strength nutrient solution consisting of H3BO3 10 µm, MnSO4 0·5 µm, ZnSO4 0·5 µm, CuSO4 0·1 µm (NH4)6Mo7O24 0·01 µm, Fe-EDTA 15 µm, KH2PO4 0·5 mm, MgSO4 1·2 mm, CaCl2 2·0 mm. Nitrogen was applied either as KNO3 and/or (NH4)2SO4 at a concentration of 2 mm N or as mentioned in the text. For NH4NO3 treatments, 1 mm K2SO4 was added to compensate for potassium applied in the KNO3 variants. Nitrogen depletion in the nutrient solution was avoided by checking the concentration in the solution at least once a day using a RQflex reflectometer (Merck, Darmstadt, Germany) and adding an appropriate amount to restore the original concentration. The nutrient solution was mixed by bubbling, the pH was held between 6·8 and 7·2 by addition of CaCO3, and the solution was changed completely every 2 d.

Plant material was harvested after 25 d under the growth light intensity and [CO2], the biomass determined, the youngest fully expanded leaf frozen in liquid nitrogen, and stored at −80 °C. Each sample point represents four different plants. At this stage, current growth is stimulated by elevated [CO2] (Geiger et al. 1998). The frozen plant tissue material was ground to a fine powder in liquid nitrogen in a mortar, and aliquots used for analyses.

Enzyme and metabolite analysis

Extracts were prepared from subaliquots of the frozen powdered leaf and root material and assayed for NIA activity as in Geiger et al. (1998) and sugars, 2-oxoglutarate, nitrate, ammonium and amino acids using coupled enzyme assays or high-performance liquid chromatography as in Geiger et al. (1998, 1999). Glutamine synthetase activity was assayed as in Scheible et al. (1997c).

Northern blot analysis

Total RNA was prepared according to the method of Logemann, Schell & Willmitzer (1987). The blot was performed as in Löw & Rausch (1994). RNA was fixed covalently to the blotting membrane (Duralon-UV membrane; Stratagene, La Jolla, CA, USA) by UV cross-linking (Stratalinker 1800; Stratagene). The blots were probed with digoxigenin-labelled NIA 2 (Vincentz & Caboche 1991), GLN1 (Dubois et al. 1996), GLN2 (Becker et al. 1992), PPC (Koizumi et al. 1991), PKc (Gottlob-McHugh et al. 1992), ICDH1 (Galvez et al., unpublished), glycine decarboxylase Subunit P-Protein (Stanislav & Bauwe 1994) NRT2 (Krapp et al. 1998), LeAMT1·2 and LeAMT2·1 (Gazzarrini et al. 1999) according to the Boehringer DIG-system, and detected on Hyperfilms-ECL (Amersham, Freiburg, Germany). The blotted membranes were routinely checked for loading by visual inspection of the rRNA under UV light.

15N Feeding experiments and nitrogen analysis

Nitrogen uptake was measured by applying 15N in the nutrient solution as K15NO3 (10% enrichment), NH415NO3 or 15NH4NO3 (20% enrichment) for 2 h and determining the 15N enrichment in the plant tissue over time. Total N uptake was calculated by multiplying the amount of 15N taken up with a correction factor for the percentage 15N supplied and divided by time and root fresh weight. 15N/14N isotopes were analysed by coupling the Dumas principle with a stable isotope mass spectrometer (Roboprep-CN and Tracermass; Europa Scientific Ltd, Crewe, UK). Total N was estimated in freeze-dried plant material with a NCS 2500 Elemental Analyzer (CE Instruments, Milan, Italy) using Dumas combustion. The sample was energetically oxidized to a gas mixture and N was determined by a thermoconductivity detector.

Results

Plant growth

Tobacco plants grew slightly faster on 1 mm ammonium nitrate than 2 mm nitrate (Fig. 1a) (see also Geiger et al. 1999). Elevated [CO2] resulted in a 2·4- and 2·8-fold stimulation of growth on 2 mm nitrate and 1 mm ammonium nitrate, respectively (Fig. 1a). The root : shoot ratio was not altered by elevated [CO2] (Fig. 1b).

Figure 1.

Biomass of tobacco growing hydroponically with 2 mm nitrate or 1 mm ammonium nitrate at ambient or elevated [CO2]. (a) Whole-plant biomass and (b) root : shoot ratio of tobacco growing in 2 mm nitrate and 400 p.p.m. [CO2] (□),2 mm nitrate and 800 p.p.m. [CO2] (▪), 1 mm ammonium nitrate and 400 p.p.m. [CO2] (□)and 1 mm ammonium nitrate and 800 p.p.m. [CO2] (▪). The plants were harvested after 25 d growth. The results are given as means ± SE (n = 4 separate plants).

NIA transcript levels and NIA activity in leaves

In plants growing on 2 mm nitrate and ambient [CO2], the leaf NIA transcript level (Fig. 2a) was high at the end of the dark period, fell rapidly after illumination, and recovered during the night. NIA activity (Fig. 3a) rose three-fold to a peak after about 4 h light, and declined during the second part of the light period and the night. Plants growing on ammonium nitrate in ambient [CO2] showed similar levels and diurnal changes of the NIA transcript (Fig. 2a) to nitrate-grown plants but had 20–30% lower NIA activity (Fig. 3b).

Figure 2.

Transcript levels in source leaves of tobacco growing hydroponically with 2 mm nitrate or 1 mm ammonium nitrate in ambient or elevated [CO2]. Leaves were harvested from plants growing in a 12 h light : 12 h dark cycle after 4, 6 or 11 h in the light and 4 or 11 h in the dark. (a) nitrate reductase (NIA); (b) plastid glutamine synthetase (GLN2); (c) cytosolic glutamine synthetase (GLN1); (d) phosphoenolpyruvate carboxylase (PPC); (e) cytosolic pyruvate kinase (PKc); and (f) glycine decarboxylase (GDC).

Figure 3.

Diurnal changes of NIA and GS activity in levels in source leaves of tobacco growing hydroponically with 2 mm nitrate or 1 mm ammonium nitrate in ambient or elevated [CO2]. Plants were grown on 2 mm nitrate (panels a, c) or 1 mm ammonium nitrate (panels b, d) at 400 (s, h) or 800 (d, j) p.p.m. [CO2] and the youngest fully developed source leaf harvested after 4, 6 or 11 h in the light and 4 or 11 h in the dark. (a) NIA activity in plants grown on nitrate and (b) NIA activity in plants grown on ammonium nitrate. (c) Total glutamine synthetase activity in plants grown on nitrate and (d) total glutamine synthetase activity in plants grown on ammonium nitrate. The results are given as means ± SE (n = 4 separate plants).

Elevated [CO2] led to a subtle modification of NIA expression in plants growing on nitrate. First, NIA transcript levels were slightly increased (Fig. 2a). This contrasts to tobacco growing on sand on high nitrate, where elevated [CO2] led to a slight decrease of the NIA transcript level (Geiger et al. 1998). The different response to elevated [CO2] may be due to the improved nitrate supply in hydroponic culture (see below). Second, although NIA activity was not altered at the midday maximum, the decline during the second part of the light period was attenuated (Fig. 3a). A similar response was found in tobacco growing in sand on high nitrate (Geiger et al. 1998).

Elevated [CO2] led to more marked changes in ammonium nitrate-grown plants. First, the absolute level of the NIA transcript was reduced (Fig. 2a). This was particularly marked at the end of the night period. Second, NIA activity was reduced, with a particularly large decrease during the first part of the light period (Fig. 3b). Third, the decline of NIA activity during the second part of the light period was attenuated (Fig. 3b).

When the results with nitrate-grown and ammonium nitrate-grown plants were combined, two separate effects of elevated [CO2] could be discerned. First, elevated [CO2] decreased the level of the NIA transcript at the end of the night (Fig. 2a) and attenuated the rise of NIA activity after illumination (Fig. 3a–b). This component was found only when ammonium was present. Second, elevated [CO2] stabilized NIA activity during the second part of the light period (Fig. 3a,b). This component was found in both nitrogen regimes. It resulted in an increase of NIA activity integrated over the entire 24 h diurnal cycle in plants growing on nitrate, but was overridden by the overall depression of NIA activity in plants growing on ammonium nitrate.

NIA transcript levels and NIA activity in roots

In the roots of plants growing with 2 mm nitrate and ambient [CO2], the NIA transcript level was lowest at the start of the light period, rose slightly at the end of the day and remained high during the night (data not shown, see also Geiger et al. 1998). The diurnal changes in the roots were small, and out of phase with those in leaves. Root NIA activity increased about two-fold during the first part of the light period (Fig. 4a, see also Geiger et al. 1998). Plants growing on ammonium nitrate in ambient [CO2] contained lower NIA transcript levels (data not shown) and slightly lower root NIA activity than nitrate-grown plants (Fig. 4b). This small decrease was confirmed in a separate batch of plants (Table 1).

Figure 4.

Diurnal changes of NIA and GS activity in roots of tobacco growing hydroponically with 2 mm nitrate or 1 mm ammonium nitrate in ambient or elevated [CO2]. Plants were grown on 2 mm nitrate (panels a, c) or 1 mm ammonium nitrate (panels b, d) at 400 (s) or 800 (d) p.p.m. [CO2] and the entire root system harvested after 4, 6 or 11 h in the light and 4 or 11 h in the dark. (a) NIA activity in plants grown on nitrate and (b) NIA activity in plants grown on ammonium nitrate. (c) total glutamine synthetase activity in plants grown on nitrate and (d) total glutamine synthetase activity in plants grown on ammonium nitrate. The results are given as means ± SE (n = 4 separate plants).

Table 1.  NIA activity (µmol g−1 FW h−1) and levels of carbohydrates, nitrate and total amino acids (µmol g−1 FW) in the roots of tobacco growing hydroponically with 2 mm nitrate or 1 mm ammonium nitrate in ambient or elevated [CO2]. All results are means ± SE (n = 4 separate plants)
  4 h light11 h light4 h dark
400 p.p.m.800 p.p.m.400 p.p.m.800 p.p.m.400 p.p.m.800 p.p.m.
Nitrate-grown plantsNIA activity11·9 ± 1·410·8 ± 0·912·8 ± 1·112·9 ± 0·710·8 ± 1·814·1 ± 1·7
Sucrose 2·2 ± 0·2 2·4 ± 0·1 2·4 ± 0·1 2·7 ± 0·1 1·5 ± 0·1 2·6 ± 0·3
Glucose 2·8 ± 0·3 3·4 ± 0·3 3·5 ± 0·5 4·4 ± 0·4 1·8 ± 0·2 3·9 ± 0·4
Fructose 3·9 ± 0·2 4·9 ± 0·4 4·5 ± 0·6 5·8 ± 0·4 3·2 ± 0·2 5·4 ± 0·6
Nitrate49·9 ± 0·751·6 ± 0·644·5 ± 2·743·9 ± 1·852·6 ± 0·951·3 ± 1·9
Total amino acids 5·1 ± 0·2 5·9 ± 0·4 5·9 ± 0·4 6·5 ± 0·210·2 ± 0·913·8 ± 1·2
Ammonium-nitrate-grown plantsNIA activity10·2 ± 0·9 8·2 ± 0·7 7·1 ± 0·4 8·7 ± 1·6 7·4 ± 0·9 5·6 ± 0·5
Sucrose 2·2 ± 0·1 2·5 ± 0·1 1·9 ± 0·1 2·6 ± 0·2 1·2 ± 0·1 2·1 ± 0·2
Glucose 2·6 ± 0·1 2·5 ± 0·1 2·2 ± 0·1 3·6 ± 0·4 1·5 ± 0·1 2·7 ± 0·2
Fructose 3·8 ± 0·2 3·9 ± 0·2 3·3 ± 0·1 5·5 ± 0·5 2·2 ± 0·3 4·4 ± 0·2
Nitrate40·9 ± 0·535·8 ± 1·432·3 ± 1·232·3 ± 1·338·2 ± 2·639·7 ± 0·8
Total amino acids 6·1 ± 0·4 5·8 ± 0·1 7·1 ± 0·7 7·2 ± 0·8 8·2 ± 1·313·2 ± 0·4

Elevated [CO2] did not consistently alter the level or the diurnal changes of the NIA transcript in the roots of nitrate-grown plants (data not shown). It led to a 20–30% decrease of root NIA activity during the day but did not alter NIA activity at night (Fig. 4a). In a separate experiment (Table 1), elevated [CO2] did not alter root NIA activity during the day and led to a slight non-significant increase of root NIA activity at night in roots of nitrate-grown plants. In earlier studies with tobacco growing on high nitrate in sand, elevated [CO2] led to an increase of root NIA activity even in the day (Geiger et al. 1998). Taken together, these studies show that elevated [CO2] has a variable effect on absolute root NIA activity in nitrate-grown plants, leading to a decrease or increase during the day, and unaltered or increased activity at night.

In ammonium nitrate-grown plants, elevated [CO2] led to a marked decrease of root NIA activity (Fig. 4b, Table 1), especially at night when NIA activity was only half that in ambient [CO2]. Elevated [CO2] therefore has contrasting effects on root NIA activity at night in nitrate-grown and ammonium nitrate-grown plants, leading to no change or an increase in the former and a decrease in the latter.

Diurnal changes of GLN2 transcript levels and glutamine synthetase activity in leaves

The GLN2 transcript was lowest at the start of the light period, and highest at the end of the light period and during the night (Fig. 2b). The levels and the diurnal changes of the transcript for plastid glutamine synthetase (GLN2) in leaves were similar in ammonium nitrate and nitrate-grown plants, and did not differ markedly between ambient and elevated [CO2].

Glutamine synthetase activity differed in the four growth conditions (Fig. 3c,d). This was mainly due to contrasting responses during the night. In ambient [CO2], glutamine synthetase activity in the light was similar in nitrate- and ammonium nitrate-grown plants, but whereas activity decreased in the dark in nitrate-grown plants it remained high in ammonium nitrate-grown plants (compare Fig. 3c,d). Elevated [CO2] tended to lead to a decrease of glutamine synthetase activity, but the response again depended on the time of day and the nitrogen source. In nitrate-grown plants, elevated [CO2] did not affect activity early in the day but led to a 20–30% decrease later in the day and during the night (Fig. 3c). A similar response to elevated [CO2] was seen in tobacco growing on sand with 12 mm nitrate (data not shown). In ammonium nitrate-grown plants, elevated [CO2] did not alter glutamine synthetase activity in the light, but led to a 50% decrease at night (Fig. 3d).

The changes of glutamine synthetase activity will include changes of the plastidic isoform GLN2, which represents about 90% of the total activity in leaves (Lam et al. 1996). The strikingly different responses of glutamine synthetase activity after darkening in the four growth conditions were not associated with changes in the level or response of the GLN2 transcript (see above). Transcript levels for cytosolic glutamine synthetase (GLN1) did not show any marked diurnal changes or differences between the four growth conditions (Fig. 2c).

Enzymes involved in carbon–nitrogen interactions were also investigated (Fig. 2). The diurnal changes of the phosphoenolpyruvate carboxylase (PPC, Fig. 2d) transcript resemble those of NIA (see also Scheible et al. 2000), whereas the diurnal changes of the transcripts for cytosolic pyruvate kinase (PKc, Fig. 2e) and NADP-isocitrate dehydrogenase (ICDH-1, data not shown) resemble those of GLN2. These diurnal changes prioritize carbon flow towards the synthesis of malate for pH regulation when nitrate is being rapidly assimilated in the first part of the light period, and towards 2-oxoglutarate synthesis later in the diurnal cycle when glutamine synthetase activity increases (see Scheible et al. 2000; Matt et al. 2001 for further discussion). Growth in nitrate or ammonium nitrate, or at ambient or elevated [CO2] did not lead to marked changes in the level or the diurnal response of the transcripts for PPC (Fig. 2d), PKc (Fig. 2e), ICDH-1 (data not shown) or the activity of PPC, PK or NADP-ICDH (data not shown). Transcript for glycine decarboxylase P-protein (Fig. 2f) was also unaffected by these treatments.

GLN1 transcript levels and GS activity in roots

In roots of nitrate-grown plants in ambient [CO2], cytosolic GLN1 transcript levels (data not shown) were highest in the first part of the light period and decreased later in the light period and at night. Root glutamine synthetase activity was also high during the day and decreased during the night (Fig. 4c). In plants growing in ammonium nitrate, the roots contained much higher levels of the GLN1 transcript and the decrease later in the light period was delayed. Elevated [CO2] led to a slight increase of the GLN1 transcript level in nitrate-grown plants, but did not lead to an additional increase of the GLN1 transcript levels in ammonium nitrate-grown plants (data not shown). Glutamine synthetase activity was similar in all four growth condtions (Fig. 4c & d).

Levels of the transcripts for nitrate and ammonium transport proteins in the roots

Figure 5 shows transcript levels for NRT2, which encodes a high affinity nitrate transporter (Krapp et al. 1998), in two separate experiments, corresponding to the plant material shown in Figs 1–4 (Fig. 5a) and Table 1 (Fig. 5b). There was a weak diurnal rhythm in nitrate-grown plants, with a maximum during the light period and a minimum at the end of the dark period. The NRT2 transcript was slightly lower in ammonium nitrate-grown plants. Elevated [CO2] did not lead to a major or consistent change of the NRT2 transcript in nitrate-grown plants. Elevated [CO2] led to a decrease of NRT2 transcript in ammonium nitrate-grown plants, except for an anomalous increase in one sample 4 h into the night (Fig. 5a).

Figure 5.

NRT2 transcript levels in roots of tobacco growing hydroponically with 2 mm nitrate or 1 mm ammonium nitrate in ambient or elevated [CO2]. The samples were prepared (a) from the plants used for the analyses of leaf metabolism (Figs 2,3,7 & 8) or (b) for the measurements of nitrate uptake (Fig. 6 and Table 1). Each sample included roots from four plants. The replicate tracks in the latter experiment show two sets of separately grown and extracted plants.

Nitrate and ammonium uptake in roots

Plants growing on 2 mm nitrate were incubated with 2 mm15N-nitrate, and plants that had been grown on 1 mm ammonium nitrate were incubated with 1 mm15N-nitrate in the presence of 1 mm unlabelled ammonium, or with 1 mm15N-ammonium in the presence of 1 mm unlabelled nitrate. The feeding experiment lasted 2 h and was carried out at three different times (2–4 h and 8–10 h into the light period, and 2–4 h into the dark period). They were carried out using the plants presented in Table 1 and Fig. 5b).

The 15N-nitrate uptake was higher during the day than at night in plants grown on nitrate in ambient [CO2](Fig. 6a). This matches the slight decrease of the NRT2 transcript in the night (see above). Nitrate uptake was decreased by 30–35% in ammonium nitrate-grown plants, compared with nitrate-grown plants (Fig. 6b, note scale of the y-axes). This matches the lower NRT2 transcript levels in ammonium nitrate-grown plants (see Fig. 5a,b). Ammonium uptake was a factor of two lower than nitrate uptake (Fig. 6c), and also decreased later in the day and at night (Fig. 6c). The overall rate of 15N-nitrate uptake plus 15N-ammonium uptake in ammonium nitrate-grown plants in ambient [CO2] (Fig. 6d) resembled the rate of 15N-nitrate uptake in nitrate-grown plants (Fig. 6a) in ambient [CO2].

Figure 6.

Rate of nitrate and ammonium uptake in tobacco growing hydroponically with 2 mm nitrate or 1 mm ammonium nitrate in ambient or elevated [CO2]. The same plants were used as those presented in Table 1. 15N was provided to hydroponically growing plants at three different times during the diurnal cycle for a 2 h period. The plants were then harvested and separated into the shoot and root, and analysed for total biomass (not shown), total N content (not shown), 15N-enrichment (not shown), and total 15N. Total uptake was calculated by summing the 15N in the root and shoot. Throughput to the shoot was estimated as the 15N in the shoot, ignoring possible recycling of 15N to the roots during the 2 h incubation. Incubation started at 2 h or 8 h into the light period or 2 h into the dark period. Incubation were carried out in growth conditions at either 400 (s) or 800 (d) p.p.m. [CO2]. (a) Uptake of 15N-nitrate by tobacco growing on 2 mm nitrate (b) Uptake of 15N-nitrate by plants growing on 1 mm ammonium nitrate. (c) Uptake of 15N-ammonium by tobacco growing on 1 mm ammonium nitrate. (d) Sum of uptake of 15N-nitrate plus 15N-ammonium in plants growing on 1 mm ammonium nitrate. The results are given as means ± SE (n = 4 separate plants).

Elevated [CO2] led to two changes. First, the diurnal response was modified, resulting in a subtle stimulation of nitrogen uptake over the entire diurnal cycle. In nitrate-grown plants, elevated [CO2] did not alter nitrate uptake in the first part of the light period, but stimulated uptake later in the light period and at night (Fig. 6a). In ammonium nitrate-grown plants, total nitrogen uptake was also unaltered in the first part of the light period and stimulated later in the light period and during the night (Fig. 6d). Second, ammonium uptake was stimulated preferentially compared to nitrate uptake. Although nitrate uptake at night was stimulated in elevated [CO2] in ammonium nitrate-grown plants, the stimulation was less marked than in nitrate-grown plants (compare Fig. 6b with Fig. 6a). It was also smaller than the stimulation of ammonium uptake (Fig. 6c), which rose from 27 to 30% of total N uptake in ambient [CO2] to 33–36% of total N uptake in elevated [CO2] (compare Fig. 6b–d).

Carbohydrates and 2-oxoglutarate in leaves

Carbon and nitrogen metabolites were investigated to provide information about their behaviour during the complex diurnal changes of transcript levels and enzyme activities. They can be used to estimate in vivo fluxes (see below), and may give information about underlying regulation mechanisms.

Elevated [CO2] led to a small increase of sucrose (Fig. 7a,b), reducing sugars (Fig. 7c,d) and starch (Fig. 7e,f). The 2-oxoglutarate levels were higher in nitrate (Fig. 7g) than ammonium nitrate-grown plants (Fig. 7h), but were not altered by elevated [CO2]. In all four conditions, 2-oxoglutarate increased about 2·5-fold after illumination, decreased in the second part of the light period, and remained low during the night. These diurnal changes are larger than in sand-grown plants (see Scheible et al. 1997c).

Figure 7.

Carbon metabolites in source leaves. The plants were grown on 2 mm nitrate (panels a, c, e, g) or 1 mm ammonium nitrate (panels b, d, f, h) in 400 p.p.m. (s) and 800 p.p.m. (d) [CO2]. (a, b) Sucrose (c, d) glucose plus fructose (e, f) starch (g, h) 2-oxoglutarate. The results are given as means ± SE (n = 4 separate plants).

Nitrate, ammonium and amino acids in leaves

Leaf nitrate (Fig. 8a) decreased during the light period and recovered at night in nitrate-grown plants. Similar diurnal changes were found in ammonium nitrate, but the absolute levels of nitrate were lower (Fig. 8b). This agrees with the lower NRT2 transcript level (Fig. 5) and the lower rate of nitrate uptake (compare Fig. 6a,b) in ammonium nitrate-grown plants. Elevated [CO2] did not markedly alter the absolute levels or the diurnal changes of leaf nitrate in nitrate-grown plants (Fig. 8a). In ammonium nitrate-grown plants, elevated [CO2] did not alter leaf nitrate levels during the day, but delayed the recovery of nitrate during the night (Fig. 8b). This is in agreement with the relatively low rate of nitrate uptake at night in ammonium nitrate-grown plants in elevated [CO2] (Fig. 6b).

Figure 8.

Nitrogen metabolites in source leaves. The plants were grown on 2 mm nitrate (panels a, c, e, g, i, k, m, o, q, s, u) or 1 mm ammonium nitrate (panels b, d, f, h, j, l, n, p, r, t, v) in 400 p. p. m. (s) and 800 p.p.m. (d) [CO2]. (a, b) nitrate (c, d) ammonium (e, f) glutamine (g, h) glutamate (i, j) glutamine:glutamate ratio (k, l) asparagine (m, n) asparatate (o, p) glycine (q, r) serine (s, t) glycine : serine ratio (u, v) total amino acids. The results are given as means ± SE (n= 4 separate plants).

Ammonium rose during the day in leaves of plants growing on nitrate (Fig. 8c, see also Scheible et al. 2000). The increase was delayed in elevated [CO2] (Fig. 8c). Far higher amounts of ammonium accumulated in plants growing on ammonium nitrate in ambient [CO2] (Fig. 8d). This accumulation was reduced but not abolished in elevated [CO2] (Fig. 8d).

Glutamine increased during the light period and decreased during the night in leaves of plants growing on nitrate in ambient [CO2] (Fig. 8e, see also Geiger et al. 1998; Scheible et al. 2000). Plants growing on ammonium nitrate and ambient [CO2] contained higher levels of glutamine, and the diurnal changes were almost completely abolished (Fig. 8f). As a result, leaf glutamine was very high at the end of the night in plants growing on ammonium nitrate (compare Fig. 8e,f). Elevated [CO2] did not markedly alter glutamine levels in nitrate-grown plants (Fig. 8e, see also Geiger et al. 1998), but led to a marked decrease of glutamine in ammonium nitrate-grown plants (Fig. 8f). The decrease was small in the first part of the light period and marked during the dark period.

Glutamate levels did not show any marked diurnal changes (Fig. 8g,h). Similar levels of glutamate were found in nitrate-and ammonium nitrate-grown plants. In elevated [CO2], there was a trend to slightly higher glutamate in the light and slightly lower glutamate in the dark, as seen previously for sand-grown plants (Geiger et al. 1998). The glutamine : glutamate ratio rose during the light period and fell during the night in plants growing on nitrate in ambient [CO2] (Fig. 8i), as seen previously for sand-grown plants (Scheible et al. 1997b, Geiger et al. 1998). Growth on ammonium nitrate led to a slightly higher glutamine : glutamate ratio during the day, and a much higher ratio in the dark (Fig. 8j). Elevated [CO2] led to a slight decrease of the glutamine : glutamate ratio during the day in nitrate-grown plants (Fig. 8i). The high glutamine : glutamate ratio at the end of the day and during the night in ammonium nitrate-grown plants was markedly lowered in elevated [CO2] (Fig. 8j).

Asparagine (Fig. 8k,l) was higher in ammonium nitrate- than in nitrate-grown plants (see also Geiger et al. 1999). Elevated [CO2] led to a slight increase of asparagine in nitrate-grown plants during the day, and a marked decrease of asparagine in ammonium nitrate-grown plants during the night. Aspartate was present at similar levels in nitrate- and ammonium nitrate-grown plants (Fig. 8m,n), and was not altered by elevated [CO2] (Fig. 8m,n).

Glycine increased during the day and decreased during the night in nitrate-grown plants in ambient [CO2] (Fig. 8o). In ammonium nitrate-grown plants in ambient [CO2], glycine did not decrease so far during the dark period and rose more rapidly at the start of the light period (Fig. 8p). These diurnal changes were strongly attenuated in elevated [CO2] in both nitrogen regimes (Fig. 8o,p). Serine levels (Fig. 8q,r) changed in a similar manner to glycine, but less dramatically. The glycine : serine ratio (Fig. 8s,t) was high during the day and decreased at night in ambient [CO2]. The glycine : serine ratio had similar values in nitrate- and ammonium-nitrate-grown plants, but was much lower in elevated [CO2] than in ambient [CO2]. The low glycine : serine ratio is a consequence of the lower rate of photorespiration. It is a robust response to elevated [CO2], and is found in nitrate and ammonium nitrate (Fig. 8s,t, also Geiger et al. 1999), and in nitrogen- replete and -deficient plants (Geiger et al. 1999).

Total leaf amino acids (Fig. 8u) increased during the day and decreased at night in plants growing in nitrate and ambient [CO2]. Similar changes were seen in ammonium nitrate and ambient [CO2] (Fig. 8v), except that the levels were higher at night due to the high glutamine at this time in ammonium nitrate-grown plants. In comparison with ambient [CO2], total amino acid levels in elevated [CO2] were unaltered or even higher in nitrate-grown plants, and similar in the light and slightly lower at night in ammonium nitrate-grown plants. The decrease at night in the latter is due to the decrease of glutamine (see Fig. 8f). In earlier studies where plants were grown with high nitrate or ammonium nitrate on sand or vermiculite (Geiger et al. 1998, 1999), there was a small decrease of leaf amino acids in elevated [CO2]. The results for the hydroponically grown plants (Fig. 8) demonstrate that the uptake and reduction of mineral nitrogen is stimulated in parallel with the acceleration of carbohydrate formation and growth in elevated [CO2], when excess nitrogen is provided.

Carbohydrates, nitrate, ammonium and amino acid levels in roots

Root sugar levels increased during the day and decreased at night in plants growing on nitrate in ambient [CO2](Fig. 9a,c, Table 1). Similar levels and diurnal changes were found ammonium nitrate-grown plants in ambient [CO2] (Fig. 9b,d, Table 1). The major effect of elevated [CO2] was to attenuate the decrease at night. Whereas root sugar levels were only marginally increased during the day they were almost two-fold higher at night in elevated [CO2], compared with ambient [CO2] (Fig. 9a–d, Table 1).

Figure 9.

Metabolite levels in roots. The plants were grown on 2 mm nitrate (panels a, c, e, g, i, k, m, o, q) or 1 mm ammonium nitrate (panels b, d, f, h, j, l, n, p, r) in 400 p. p. m. (s) and 800 p.p.m. (d) [CO2]. (a, b) Sucrose (c, d) glucose plus fructose (e, f) nitrate (g, h), glutamine (i, j) glutamate (k, l) asparagine (m, n) asparatate (o, p), glycine and (q, r) serine. The results are given as means ± SE (n= 4 separate plants).

Root nitrate levels were high (Fig. 9e,f, Table 1) and, in contrast to leaves, did not show marked diurnal changes. Nitrate was lower in ammonium nitrate-grown plants. Elevated [CO2] led to a slight increase (Fig. 9f) or did not alter (Table 1) root nitrate in nitrate-grown plants, and did not alter root nitrate in ammonium nitrate-grown plants (Fig. 9f, Table 1).

Glutamine did not show any marked diurnal changes in the roots of nitrate-grown plants in ambient [CO2] (Fig. 9g). Plants growing in ammonium nitrate contained higher levels of glutamine in their roots, especially at night (Fig. 9h). Elevated [CO2] led to an increase of root glutamine at the end of the day and in the night in nitrate-grown plants (Fig. 9g) and an increase at night in ammonium nitrate-grown plants (Fig. 9h). Glutamate was slightly lower in ammonium nitrate- than nitrate-grown plants, and did not show marked diurnal changes (Fig. 9i,j). Elevated [CO2] led to a slight increase of glutamate in the night, especially in nitrate-grown plants (Fig. 9i). Asparagine was present at similar levels in the light and increased slightly in the dark. There were no consistent changes between the four treatments (Fig. 9k,l). Aspartate was slightly higher in nitrate- (Fig. 9m) than in ammonium nitrate-grown plants (Fig. 9n). Elevated [CO2] led to a small increase of aspartate in the dark (Fig. 9m,n). Glycine (Fig. 9o,p) and serine (Fig. 9q,r) levels were similar in nitrate- and ammonium nitrate-grown plants and did not show any consistent changes in elevated [CO2] (Fig. 9o–r). Taken together, a trend emerges in which elevated [CO2] has little effect during the day but leads to a slight increase of root amino acid levels during the night. This was confirmed for the plants used for transport measurements (Table 1).

Comparison of uptake rates, diurnal changes of metabolites pools and enzyme activities

The rate of nitrate reduction in leaves at different times of the diurnal cycle was estimated by comparing the diurnal changes of nitrate and ammonium uptake with the diurnal changes of nitrate and other nitrogen-containing metabolites (see the legend to Table 2 for the details and Matt et al. 2001 for discussion of the assumptions involved). This approach was used because calculations based on the metabolism of 15N are subject to large errors, due to changes in enrichment when 15N-nitrate mixes with the large pools of 14N-nitrate in the plant. To carry out the calculation, all of the results are expressed on a whole plant basis. The rates of nitrate and ammonium uptake during the day and the night are estimated assuming that the rate of uptake between 2 and 4 h and 8–10 h into the day are typical for the light period, and the rate between 2 and 4 h into the night is typical for the night. The amplitude of the diurnal changes of the leaf nitrate pool are estimated as the difference between the maximum and minimum nitrate content, which in almost all of the treatments corresponds to the end of the night and the end of the day, respectively (see Fig. 8a,b).

Table 2.  Quantitative comparison of nitrate and ammonium uptake rates, diurnal changes of the pools of nitrate and the proximal products of nitrate assimilation, and in vitro NIA and glutamine synthetase activity. Nitrate uptake is calculated from Fig. 6a–b,ammonium uptake from Fig. 6c, the diurnal changes of leaf nitrate from Fig. 8c, the diurnal changes of nitrogen accumulated in the proximal products of nitrate assimilation (ammonium, glutamine plus glycine and serine) from Fig. 8c–d, 8d–e, 8o–p and 8q–r, leaf NIA activity from Fig. 2a–b, leaf glutamine synthetase activity from Fig. 2c–d, root NIA activity from Fig. 4a–b, and root glutamine synthetase activity from Fig. 4c–d. Diurnal changes of nitrate in leaves, and the diurnal changes of nitrogen accumulated in the proximal products of nitrate assimilation (ammonium, glutamine, glycine, serine) are estimated as the difference between the maximum and minimum content. All results are recalculated on a whole plant basis to allow uptake rates to be compared with changes of pools in the source leaves and roots. To do this, data for roots (expressed in Figs 4, 6 and 9 on a root fresh weight basis) is multiplied by the root weight ratio (0·27 and 0·29 for nitrate-and ammonium nitrate-grown plants, see Fig. 1), and data for source leaves (expressed in Figs 2 and 8 on a leaf fresh weight basis) by an estimated leaf weight ratio of 0·58
ParameterAmbient [CO2]Elevated [CO2]
NitrateAmmonium nitrateNitrateAmmonium nitrate
Fluxes or activities (µmol g−1 plant FW h−1):
Nitrate uptake, day and night4·5–3·23·2–2·14·2–4·83·0–2·6
Ammonium uptake, day and night 1·3–0·8 1·6–1·5
Total nitrogen uptake4·5–3·24·4–2·94·2–4·84·6 4·1
Rate of depletion of leaf nitrate during the 
first 6 h of the photoperiod
4·85·16·72·9
Estimated rate of nitrate assimilation10·3–08·3–010·9–05·9–0
Diurnal volume (µmol g−1 plant FW 24 h−1):
Total nitrogen uptake during one diurnal cycle9288108.104
Total nitrate uptake during one diurnal cycle9253108.67
Diurnal change of leaf nitrate38364025
Diurnal change of nitrogen in the proximal 
intermediates of nitrate assimilation in the leaf
16101310
In vitro activities (µmol g−1 plant FW h−1):
Leaf NIA activity, midday and night18–4·415–3·618–3·610–3·4
Leaf GS activity, day and end of night55–9994–8144–9332–96
Root NIA activity, midday and night4·9–2·44·1–1·64·5–2·53·8–0·7
Root GS activity, midday and night20–1119–1317–1016–8

Table 2 also investigates the balance between nitrate and ammonium metabolism. The diurnal changes of nitrogen in ammonium, glutamine, glycine and serine in leaves (Figs 8c–f,o–r) are summed to estimate the amount of reduced nitrogen that accumulates temporarily in the proximal products of nitrogen assimilation. Ammonium is included even in plants growing on ammonium nitrate on the assumption that most of the ammonium entering the plant is assimilated in the roots, and that ammonium in the leaves derives from nitrate assimilation or photorespiration.

The results for the reference treatment (nitrate-grown plants in ambient [CO2]) resemble those presented for a separate experiment in Matt et al. (2001). The diurnal changes of the leaf nitrate pool (38 µmol nitrate g−1 plant FW) and the immediate downstream products (16 µmol reduced N g−1 plant FW) are large compared to the total daily influx of nitrate into the plant (92 µmol nitrate g−1 plant FW), revealing that there are major transient imbalances between nitrate uptake, nitrate reduction and ammonium assimilation. During the first 6 h of the light period the rate of nitrate assimilation in leaves (> 9 µmol nitrate g−1 plant FW) is two-fold higher than the rate of nitrate uptake, leading to a rapid decline of leaf nitrate. The amount of nitrogen that accumulates in ammonium, glutamine, glycine and serine during the light period represents 18% of the overall amount of nitrate that enters the plant in a 24 h cycle. The decline of nitrate and the accumulation of downstream products is reversed later in the diurnal cycle, especially at night when nitrate assimilation is very low. The absorbed nitrate is used to refurbish the leaf nitrate pool, and nitrogen that has accumulated in ammonium, glutamine, glycine and serine is remobilized. These dramatic changes in fluxes and metabolite pools mirror the marked changes in the relation between NIA activity, nitrate uptake and GS activity during the 24 h diurnal cycle (see Figs 3 & 6, also summarized in Table 2).

Even though plants growing on ammonium nitrate take up 40% less nitrate per day and contain smaller overall pools of nitrate, the amplitude of the diurnal changes in leaves is similar to that of nitrate-grown plants. The estimated rate of nitrate assimilation during the first part of the light period in ammonium-nitrate-grown plants is 160% higher than the rate of nitrate uptake, compared with 117% in nitrate-grown plants. This is in agreement with the relatively small decrease of in vitro NIA activity (16%) compared with the decrease of nitrate uptake (approximately 30%) in ammonium nitrate-plants compared with nitrate-grown plants. Diurnal changes of the leaf nitrate pool therefore make an even larger contribution to the daily nitrate economy in ammonium nitrate-than in nitrate-grown plants. The imbalance between nitrate reduction and ammonium metabolism, on the other hand, is less marked in ammonium nitrate- than in nitrate-grown plants. About 30% less nitrogen accumulates in the proximal products of nitrate assimilation in the leaves of ammonium nitrate- than in nitrate-grown plants in ambient [CO2]. The decrease would be even larger, if some of the ammonium or glutamine is imported from the roots. Decreased accumulation of downstream products is consistent with the finding that leaves of ammonium nitrate-grown plants have lower NIA activity and lower rates of nitrate assimilation than nitrate-grown plants, whereas leaf glutamine synthetase activity is similar to or higher than in nitrate-grown plants.

In plants growing on nitrate, elevated [CO2] led to a 17% increase of nitrate uptake on a daily basis, due to increased uptake at night (Table 2). The decline of leaf nitrate during the first part of the light period resembled that in ambient [CO2], both in absolute terms and relative to the total nitrate taken up and assimilated per day (37% in elevated [CO2] compared with 41% in ambient [CO2]). In agreement, the nitrate uptake rate and NIA activity during the first part of the light period are similar in ambient and elevated [CO2]. Slightly less nitrogen accumulates in the proximal products of nitrate assimilation in elevated [CO2], both in absolute terms and when it is related to the amount of nitrate that is taken up and assimilated each day (12% in elevated [CO2] compared to 17% in ambient [CO2]). NIA activity rises slightly compared to glutamine synthetase activity, indicating that the decreased accumulation of downstream products is not due to changes in the balance between de novo nitrate and ammonium assimilation. It correlates with a lower glycine : serine ratio (see Fig. 8s,t), indicating that the lower rate of photorespiration allows better metabolism of the products of nitrate assimilation.

In plants growing on ammonium nitrate, elevated [CO2] leads to a lower rate of nitrate uptake, smaller diurnal changes of the leaf nitrate pool, and a lower rate of mobilization of nitrate in the first 6 h of the light period (Table 2). The estimated rate of nitrate assimilation in the first 6 h of the light period is 30% lower than in plants growing in ammonium nitrate and ambient [CO2], and 50% lower than in plants growing in nitrate and elevated [CO2]. This inhibition of nitrate assimilation correlates with a marked reduction of NIA activity. Glutamine synthetase activity remains high, especially during the day which, together with the lower rate of photorespiration, may explain why downstream products do not accumulate in leaves of plants growing in ammonium nitrate and elevated [CO2].

Discussion

Dynamic diurnal regulation of nitrogen metabolism provides a buffering network to stabilize the long-term rate of nitrate uptake and assimilation

The results in this and earlier papers allow several general conclusions about the operation and significance of a network that regulates nitrate uptake and assimilation. First, this network generates dramatic and dynamic changes during the diurnal cycle. These include large changes in the expression of genes required for the uptake (Gojon et al. 1998; von Wiren et al. 2000a, b, Fig. 5) and assimilation of nitrate (Scheible et al. 1997b; Stitt & Krapp 1999) and ammonium (Matt et al. 2001, Fig. 2) and genes that are required for related events in carbon metabolism (Scheible et al. 2000), significant changes in the activities of the corresponding proteins (Scheible et al. 1997b, 2000), and large changes in the levels of key intermediates and metabolic fluxes (Scheible et al. 1997b, c, 2000; Geiger et al. 1998; Matt et al. 2001). Second, the overall rate of mineral nitrogen uptake and assimilation is insensitive to changes in the nitrogen source, because the uptake and assimilation of ammonium leads to a compensating decrease in the rate of nitrate uptake and assimilation. We have shown previously that it is also insensitive to changes in the number of functional NIA gene copies (Scheible et al. 1997b, c). Third, the rate of nitrate and/or ammonium uptake and assimilation rises in parallel with the increased rate of photosynthesis in elevated [CO2], provided enough mineral nitrogen is available. Fourth, the adjustment and stabilization of nitrogen metabolism to changed conditions almost always involves a modification of the diurnal changes. This network therefore allows dramatic changes in response to sudden changes of conditions, but in the long term acts as a buffer that stabilizes the overall rate of nitrogen assimilation and coordinates it with the utilization of nitrogen for growth.

A key element in this network is the excess capacity for nitrate assimilation in wild-type tobacco growing in favourable light regimes and high nitrate. This capacity is partially or strongly inhibited for much of the diurnal cycle (Matt et al. 2001 and Table 2). During the first part of the light period maximal rates of nitrate assimilation are briefly achieved, and the rate of nitrate assimilation is much higher than the rates of nitrate uptake and ammonium metabolism. This leads to depletion of leaf nitrate and accumulation of downstream products including ammonium, glutamine and the photorespiratory metabolites glycine and serine. Later in the light period and during the night NIA activity declines, whereas nitrate uptake remains relatively high and glutamine synthetase activity rises. This facilitates recovery of the leaf nitrate pool and remobilization of accumulated downstream products. In mutants with a decreased number of functional NIA gene copies, the decrease of NIA activity is delayed, allowing the lower rate of nitrate assimilation in the first part of the light period to be compensated by a higher rate of nitrate assimilation later in the diurnal cycle (Scheible et al. 1997b). The following discussion summarizes how changes in the diurnal regulation of nitrate uptake and metabolism allow the uptake and assimilation of mineral nitrogen to be adjusted when less nitrate is required because ammonium is present in the medium or when more nitrogen is required because growth is stimulated by elevated [CO2], and then discusses some implications of our results for the underlying regulation mechanisms.

Changes in diurnal metabolism in plants growing on ammonium nitrate

The key element in the long-term response to ammonium is a partial inhibition of nitrate utilization. This inhibition is stoichiometrically balanced with ammonium uptake and assimilation, and involves coordinated changes in nitrate uptake, nitrate reduction and ammonium assimilation in the roots and leaves of the plant.

In comparison with plants growing on 2 mm nitrate, roots of plants growing on 1 mm ammonium nitrate had lower levels of the NRT2 transcript, 30–35% lower rates of nitrate uptake, and contained less nitrate. The roots also had slightly lower levels of the transcript for NIA and lower NIA activity. The lower rate of nitrate uptake was stoichiometrically balanced by the uptake of ammonium. Although the GLN1 transcript level increased, there was no significant change of root glutamine synthetase activity, indicating that ammonium is assimilated using the capacity released by the lower rate of nitrate uptake and assimilation. The levels of amino acids in the roots were similar to or slightly higher than in nitrate-grown plants.

In leaves, nitrate levels were decreased and NIA activity was consistently 20–25% lower than in nitrate-grown plants. The timing and amplitude of the diurnal changes of nitrate, the levels and the diurnal changes of the NIA transcript and the timing of the diurnal changes of NIA activity were nevertheless identical to those in nitrate-grown plants. As a result, the imbalance between nitrate import and nitrate assimilation was even more marked than in the leaves of nitrate-grown plants. Ammonium and glutamine also accumulated to higher levels than in plants growing on nitrate, and glutamine remained high during the night rather than falling as in plants growing on nitrate alone. These high pools presumably reflect the assimilation of ammonium (see next paragraph). It is intriguing (see below for further discussion) that the diurnal changes of NIA expression are retained even though ammonium and glutamine show a very different response to that seen in nitrate-grown plants, because it is widely assumed (see Stitt & Krapp 1999) that these downstream metabolites play a key role in regulating NIA expression.

Growth of plants on ammonium nitrate did not alter transcript levels for GLN1 and GLN2 in leaves, or the activity of glutamine synthetase in the light. This is not unexpected, because ammonium assimilation in leaves is dominated by fluxes of photorespired ammonium (see Matt et al. 2001). The rate of ammonium assimilation in leaves may even decrease when plants are grown on ammonium nitrate instead of nitrate, because less ammonium will be formed during nitrate assimilation in the leaves (see above) and most of the ammonium absorbed from the medium may be assimilated in the roots and exported as amino acids to the leaves (Forde & Clarkson 1999). The increase of ammonium in the leaves nevertheless indicates that some ammonium is transported to the leaves. The most striking change compared to nitrate-grown plants is that glutamine synthetase activity remains high during the night in ammonium nitrate-grown plants, whereas it decreases in nitrate-grown plants. This may facilitate metabolism of the ammonium that accumulates during the day in leaves of plants growing on ammonium nitrate, and could explain why glutamine levels remain high throughout the night. Intriguingly, the high glutamine synthetase activity at night in ammonium nitrate-grown plants was not accompanied by any change of GLN1 and GLN2 transcript levels (see below for further discussion).

Elevated [CO2] leads to coordinate changes in the diurnal regulation of nitrate uptake in the roots and nitrate reductase activity in the leaves, which facilitate uptake and assimilation later in the diurnal cycle

In nitrate-grown plants, elevated [CO2] leads to a co-ordinated modification of the diurnal changes of nitrate uptake and assimilation. In roots, nitrate uptake increases at night instead of decreasing as it does in ambient [CO2]. In leaves, the levels of nitrate, the diurnal changes of the NIA transcript level, and NIA activity at the diurnal maximum at midday resemble those in ambient [CO2], but the decline of NIA activity in the second part of the light period is attenuated. This coordinated relaxation of the feedback regulation of nitrate uptake and nitrate assimilation allows maximum rates to be maintained for a larger part of the diurnal cycle in elevated [CO2], increasing the integrated overall rate of nitrate uptake and assimilation, and explaining how amino acids and (see Geiger et al. 1999) protein are maintained at levels that are similar to or even higher than those in ambient [CO2].

Elevated [CO2] preferentially stimulates ammonium uptake and inhibits NIA expression in the roots and leaves of plants growing on ammonium nitrate

Elevated [CO2] had two separate effects on nitrogen uptake in plants growing on ammonium nitrate. First, as already seen for plants growing on nitrate, nitrogen uptake was stimulated at night. Second, there was a preferential stimulation of ammonium uptake compared with nitrate uptake. Although elevated [CO2] led to a slight increase of nitrate uptake at night in plants growing on ammonium nitrate, the stimulation was small compared with the stimulation of ammonium uptake, and was also small compared with the stimulation of nitrate uptake by elevated [CO2] in nitrate-grown plants (see above). Elevated [CO2] also led to a decrease of the NRT2 transcript for most of the diurnal period in ammonium nitrate-grown plants.

Elevated [CO2] did not alter glutamine synthetase expression in the roots of plants growing in ammonium nitrate but did lead to a decrease of NIA activity, especially during the night when ammonium uptake was most markedly stimulated. This contrasts to plants growing with nitrate, where NIA activity stayed unaltered or increased slightly at night in elevated [CO2] (see Results and Geiger et al. 1999). The lower rate of nitrate uptake and the lower NIA activity in the roots of ammonium nitrate-grown plants in elevated [CO2] presumably releases GOGAT pathway capacity for de novo ammonium assimilation in the roots. The increase of glutamine and other amino acids during the night in elevated [CO2] in the roots of plants growing on ammonium nitrate is consistent with an increased rate of ammonium assimilation.

Elevated [CO2] led to a marked decrease of NIA activity in the leaves of ammonium nitrate-grown plants. This response included two antagonistic components. First, elevated [CO2] interfered with the recovery of the NIA transcript level during the night, and inhibited the increase of NIA activity after illumination. A low level of the NIA transcript at the end of the night and an attenuation of the light-dependent increase of NIA protein and activity is also found in tobacco in short days (see Matt et al. 1998). Presumably, in both conditions NIA transcript levels at the end of the night are too low to support a rapid increase of NIA activity after illumination. The reason for the low NIA transcript level at the end of the night, however, is different in these two conditions. Whereas low sugar is responsible for the low transcript in short day-grown plants (Matt et al. 1998; Klein et al. 2000), sugars rise in elevated [CO2] and (see below) the key factor may be the lower rate of nitrate uptake and the resulting delay in replenishing nitrate during the night. Second, elevated [CO2] attenuated the decline of NIA activity in the second part of the light period. This resembles the response to elevated [CO2] in nitrate-grown plants, and indicates that some factor prevailing later in the day in elevated [CO2] in both nutrition regimes counteracts the diurnal decline of NIA activity (see below for further discussion). The net result in ammonium nitrate-grown plants is nevertheless a 50% inhibition of NIA activity and nitrate reduction over the entire 24 h cycle.

Lower rates of photorespiration and lower NIA activity presumably reduce ammonium formation in the leaf, leading to much less accumulation of ammonium during the light period than in plants growing on ammonium nitrate in ambient [CO2]. Elevated [CO2] had little effect on the levels of GLN2 or GLN1 transcript in leaves, and the glutamine synthetase activity remained high during the light period, although it did decrease in the dark.

Possible mechanisms underlying the changes in the diurnal regulation of nitrate uptake and metabolism in the roots

Feeding experiments and studies of transformants with low NIA activity indicate that NRT2 is induced by nitrate and sugars, and repressed by ammonium, glutamine or related downstream products of nitrate assimilation (Krapp et al. 1998; Gojon et al. 1998). Diurnal changes of transcripts for a set of high and low affinity nitrate transporters in tomato also indicate that their expression responds to changes of sugars, nitrate and nitrogen metabolites (Ono, Frommer & von Wiren 2000). In our treatments, root nitrate was high throughout the day and night. The decrease of NRT2 expression and the lower rates of nitrate uptake were accompanied by an increase of glutamine and asparagine in the roots of ammonium nitrate-grown plants, compared with nitrate-grown plants. The higher rates of nitrate uptake rates at night in plants growing in elevated [CO2] correlated with higher levels of sugars in the roots at night, compared with plants growing in ambient [CO2].

The stimulation of ammonium uptake at night in elevated [CO2] correlated with higher levels of root sugars at night, but was not correlated with changes of nitrate, glutamine or other amino acids. Expression of three Arabidopsis high-affinity ammonium transporters is regulated by the nitrogen supply to the roots (Gazzarrini et al. 1999). In tomato, LeAMT1·2 was induced by nitrate or ammonium, whereas LeAMT1·1 increased in nitrogen-deficient plants (von Wirén et al. 2000a). The stimulation of ammonium uptake at night in elevated [CO2] was not accompanied by marked changes in the levels of transcripts detected using LeAMT1·1 and LeAMT1·2 were used as probes with our tobacco root samples (N. von Wiren, P. Matt, data not shown). This may be because the stimulation involves changes of specific members of the AMT family, which cannot be detected using heterologous sequences from tomato, or because elevated [CO2] acts via post-transcriptional mechanisms.

Root NIA activity showed a variable response to elevated [CO2] (see Results). This may be related to different responses of root sugar levels. Root sugar levels showed a rather small increase that was mainly restricted to the night in the present experiments, and NIA activity tended to rise at night but not in the day. In the experiments of Geiger et al. (1998) with sand-grown tobacco, elevated [CO2] led to an increase of sugars in the day as well as the night, and root NIA activity showed a small increase during the day as well as a large increase at night. The decrease of root NIA activity in ammonium nitrate was correlated with lower levels of nitrate and slightly higher levels of amino acids.

Possible mechanisms underlying the diurnal regulation of leaf nitrate metabolism

NIA expression is induced by nitrate, decreased by glutamine or related compounds, and increased by sugars (Stitt & Krapp 1999). The diurnal changes of the NIA transcript or NIA activity were not correlated with the diurnal changes of leaf sugars. Leaf sugars have to fall to very low levels to exert a marked effect on NIA expression (Matt et al. 1998; Klein et al. 2000), and sugars remain above these critical levels in the conditions investigated in the present study. The diurnal changes of the NIA transcript and glutamine in leaves were inversely related in nitrate-grown plants, as seen in earlier studies (see Introduction). However, this relation breaks down in the other three growth conditions. When plants growing on ammonium nitrate and nitrate are compared in ambient [CO2], leaves of the former contain higher glutamine and the diurnal changes of glutamine are almost abolished, but the absolute levels and the diurnal changes of the NIA transcript level are identical in both sets of plants. When plants growing on ammonium nitrate are compared in ambient and elevated [CO2], the latter contain much lower glutamine but NIA transcript is also decreased. These results are inconsistent with a major or general role for glutamine in the diurnal regulation of NIA expression in leaves. They are also inconsistent with our initial hypothesis, which was that elevated [CO2] represses NIA in ammonium nitrate-grown plants because it leads to a higher rate of ammonium uptake and assimilation and a larger pool of glutamine. These conclusions are, of course, based on changes of total leaf glutamine, and could be in error if a small discrete pool is sensed.

The diurnal changes of the NIA transcript were positively correlated with the diurnal changes of leaf nitrate. Both parameters decreased rapidly during the first part of the day and recovered during the night. When all four growth treatments are compared, there is still a reasonable match between leaf nitrate levels and NIA transcript levels. In particular, nitrate levels are low at night in the leaves of plants growing on ammonium nitrate in elevated [CO2], where the recovery of the NIA transcript is suppressed. These results indicate that nitrate makes an important contribution to the diurnal regulation of NIA expression. It is unlikely, however, that the signal is related to the total leaf nitrate pool. Most of the nitrate is located in the vacuole (Miller & Smith 1996). Instead, it appears that NIA expression responds to changes in the balance between the nitrate delivery and nitrate assimilation. Earlier studies in cell suspension cultures (Heimer & Filner 1971), in maize plants and detached maize leaves (Shaner & Boyer 1975a, 1975b) and tobacco plants (Geiger et al. 1998) indicated that NIA expression responds to changes in the supply rather than the overall concentration of nitrate. It appears plausible that this results in significant changes of an intermediate pool, possibly cytoplasmic nitrate, which regulates NIA expression. A signal related to the balance between nitrate influx into the leaf and nitrate utilization is also required to trigger the massive changes in flux across the tonoplast membrane revealed by the calculations in Table 2.

An additional level of complexity is introduced because NIA activity often changes independently of the NIA transcript level. One example is the two- to three-fold increase of NIA activity after illumination, when the NIA transcript is already falling. This is due to increased synthesis and decreased degradation of NIA after illumination (Weiner & Kaiser 1999). Another example is the 30% decrease of NIA activity in ammonium nitrate-grown plants compared to nitrate-grown plants in ambient [CO2] (see Fig. 3c & d), even though NIA transcript levels are similar in both conditions (Fig. 2). This decrease is associated with high leaf glutamine (compare Figs 8e and 8f), and can be simulated by supplying 20 mm glutamine for 4 h to the roots of intact nitrate-grown plants (data not shown). A third example relates to the decrease of NIA activity during the second part of the light period. Even though elevated [CO2] does not prevent the dramatic decrease of the NIA transcript level, it does delay the decrease of NIA activity (Fig. 2a,b and Geiger et al. 1998). In this case, stabilization of NIA activity is again accompanied by a marked decrease of the leaf glutamine levels. These results indicate (see also Geiger et al. 1998) that high glutamine leads to a decrease of NIA activity via post-transcriptional or post-translational mechanisms.

Possible mechanisms underlying the diurnal regulation of ammonium metabolism

Elevated [CO2] did not alter the level or the diurnal changes of the GLN2 or GLN1 transcripts in leaves, and had no marked effect on glycine decarboxylase expression. This resembles earlier studies in tobacco at 2000 p.p.m. (Migge et al. 1997), but differs from studies in tomato where elevated (800 p.p.m.) [CO2] led to a small decrease of the transcripts for GLN1, GLN2 and SHMT (encoding the serine hydroxymethyl transferase subunit of glycine decarboxylase) in tomato leaves (von Wiren et al. 2000a).

Glutamine synthetase activity was affected by changes in the nitrogen source and [CO2], in particular during the night. After darkening, glutamine synthetase activity decreased slightly in nitrate-grown plants in ambient [CO2], decreased more markedly in nitrate-grown plants in elevated [CO2], rose in ammonium nitrate-grown plants in ambient [CO2], and decreased markedly in ammonium nitrate-grown plants in elevated [CO2]. These changes are not related to the rate of ammonium uptake. For example, when ammonium nitrate-grown plants are compared in ambient and elevated [CO2], there is an inverse relation between leaf glutamine synthetase activity in the dark and the rate of ammonium uptake. This is hardly surprising, as most of the absorbed ammonium is assimilated in the root. There is a strong correlation between leaf ammonium levels at the end of the day and the maintenance of high glutamine synthetase activity in the dark. The changes in glutamine synthetase activity in the dark must include changes in GLN2 activity (see Results). As neither the GLN2 nor the GLN1 transcript level varied between the growth conditions or decreased in the first part of the night, our results indicate that high ammonium or a related metabolite may act either post-transcriptionally or post-translationally to stabilize glutamine synthetase activity after darkening.

In conclusion, a complex regulatory network continuously adjusts the expression and activity of many proteins involved directly and indirectly in the uptake and assimilation of nitrate and ammonium. This network allows rapid changes in response to changes in the external conditions or the internal physiological status, and acts over longer time periods to stabilize the overall rate of nitrogen assimilation and coordinate it with the rate of growth. Wild-type plants growing in a favourable light regime on high nitrate possess excess capacity for nitrate assimilation, which is down-regulated for much of the light cycle to allow a long-term balance to be established with the rate of nitrate uptake and the rate of ammonium utilization. When ammonium is present in the growth medium, the rate of nitrate uptake and assimilation is further reduced to balance the nitrogen that is derived from ammonium. In elevated [CO2], the overall rate of nitrogen uptake and assimilation is increased by a modification of the diurnal changes that allows the maximum rates of nitrate uptake and assimilation to be maintained for a larger part of the diurnal cycle in nitrate-grown plants, whereas in ammonium-nitrate-grown plants there is also a preferential stimulation of ammonium uptake and assimilation. With respect to the underlying mechanisms, the data matrix we have collected indicates that changes of root sugar and amino acid levels regulate nitrate and ammonium uptake, that changes in the balance between nitrate influx and utilization play a central role in the diurnal regulation of NIA transcript levels in leaves, that high glutamine acts via post-transcriptional mechanisms to decrease NIA activity, and that high ammonium acts via post-transcriptional mechanisms to stimulate glutamine synthetase activity.

Acknowledgments

This research was supported by the Deutsche Forschungsgemeinschaft (Sti78/2–3). We are grateful to Nico von Wiren for discussions and constructive criticisms of the manuscript.

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