JXB Advance Access originally published online on December 13, 2004
Journal of Experimental Botany 2005 56(410):309-321; doi:10.1093/jxb/eri059
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RESEARCH PAPER |
Metabolic profiling reveals altered nitrogen nutrient regimes have diverse effects on the metabolism of hydroponically-grown tomato (Solanum lycopersicum) plants
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Golm, Germany
* To whom correspondence should be addressed. Fax: +49 331 5678408. E-mail: fernie{at}mpimp-golm.mpg.de
Received 25 June 2004; Accepted 22 October 2004
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Abstract |
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The role of inorganic nitrogen assimilation in the production of amino acids is one of the most important biochemical processes in plants. For this reason, a detailed broad-range characterization of the metabolic response of tomato (Solanum lycopersicum) leaves to the alteration of nitrate level was performed. Tomato plants were grown hydroponically in liquid culture under three different nitrate regimes: saturated (8 mM
), replete (4 mM ) and deficient (0.4 mM ). All treatments were performed under varied light intensity, with leaf samples being collected after 7, 14, and 21 d. In addition, the short-term response (after 1, 24, 48, and 94 h) to varying nutrient status was evaluated at the higher light intensity. GC-MS analysis of the levels of amino acids, tricarboxylic acid cycle intermediates, sugars, sugar alcohols, and representative compounds of secondary metabolism revealed substantial changes under the various growth regimes applied. The data presented here suggest that nitrate nutrition has wide-ranging effects on plant leaf metabolism with nitrate deficiency resulting in decreases in many amino and organic acids and increases in the level of several carbohydrates and phosphoesters, as well as a handful of secondary metabolites. These results are compared with previously reported transcript profiles of altered nitrogen regimes and discussed within the context of current models of carbon nitrogen interaction. Key words: Amino acids, carbon metabolism, light intensity, nitrate reduction, nitrogen metabolism, organic acids, Solanum lycopersicum, tomato
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Introduction |
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Nitrate is a major source of nitrogen for plants; indeed, most plants devote a significant portion of their carbon and energy reserves to its uptake and assimilation. In plants, nitrate has been demonstrated to serve both as nutrient and signal metabolite and as such has profound effects on both plant metabolism and growth (Glass, 2003
; Raven, 2003; Stitt, 1999; Coruzzi and Zhou, 2001; Forde, 2002; Orsel et al., 2002). Plants have evolved complex mechanisms to detect nitrate and to integrate its assimilation with photosynthesis and the overall metabolism of nitrogen and carbon (Bowsher et al., 1989, 1992). In order to deal with this complexity nitrate uptake, and its subsequent metabolism are highly regulated in plants (Stitt et al., 2002). Following its uptake from the soil, via specific transporters (Wang et al., 1998; Guo et al., 2001), nitrate is reduced to ammonium by the concerted action of nitrate reductase (NR: Foyer and Ferrario, 1994; Foyer et al., 1998), and nitrite reductase (NiR: Bowsher et al., 1988; Hoff et al., 1994). Ammonium is then primarily incorporated into amino acids by the reactions catalysed by glutamine synthetase (GS) and glutamate synthase (GOGAT: Ferrario-Mery et al., 2002; Lancien et al., 2002).
The synthesis of organic acids, especially 2-oxoglutarate, which acts as the acceptor for ammonium in the GOGAT pathway, and malate which acts as a counter-anion and substitutes for nitrate to prevent alkalization, is essential for nitrate assimilation (Stitt, 1999). In this context, it is intriguing that nitrate levels can reprogramme carbohydrate metabolism: previously it was shown that, during nitrate assimilation, carbohydrate synthesis is decreased and more carbon is converted to organic acids (Stitt et al., 2002). It was first demonstrated by Scheible et al. (1997) that nitrate supply led to a marked increase of transcript level and enzyme activity of phosphoenolpyruvate carboxylase, pyruvate kinase, citrate synthase, and isocitrate dehydrogenase and, consequently, to the accumulation of malate and 2-oxoglutarate. These studies, and other early studies on the role of nitrate as a signal metabolite (reviewed in Crawford, 1995) have recently been augmented by the publication of large-scale transcriptomic data sets on the response of both Arabidopsis (Wang et al., 2000, 2003) and tomato (Wang et al., 2001) to varying nitrate nutrition. In addition to the known nitrate-responsive genes described above, genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes of glycolysis, of trehalose-6-phosphate metabolism, iron transport/metabolism and of sulphate uptake/reduction. In many cases, only a few selected genes of small gene families were induced by nitrate, suggesting that the effect of nitrate on gene expression is substantial yet selective and affects many genes involved in carbon and nutrient metabolism. In addition, the response of key metabolic intermediates has been determined in many cases wherein nitrate assimilation has been modified by genetic or environmental perturbation (Matt et al., 2001a, b; Muller et al., 2001; Masclaux-Daubresse et al., 2002). However, to date studies of the broader range of metabolites, such as those achievable by metabolite profiling (Fernie et al., 2004a; Stitt and Fernie, 2003; Sumner et al., 2003), have not been attempted in order to characterize the response to altered nitrogen nutrition. In the present study, tomato wild-type plants were grown hydroponically on liquid culture under different nitrate and light regimes. The levels of a wide range of metabolites were analysed from leaf material harvested from these plants using gas chromatography-mass spectrometry. The results will be compared and contrasted to previously published transcriptomic data sets that profile nitrogen stress in tomato and Arabidopsis and with studies evaluating key metabolite levels in tobacco. Finally, the data will be discussed in the context of current models of nitrate assimilation in plants.
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Materials and methods |
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Plant material and growth of plants
Tomato seeds (Solamum lycopersicum cv. Moneymaker) were germinated on Murashige and Skoog media (Murashige and Skoog, 1962
) containing 2% (w/v) sucrose and were grown in a growth chamber under a light intensity of 500 µmol photons m–2 s–1, at 25 °C under a 12/12 h light/dark regime for the time interval described in the text. Subsequently, seedlings were transferred to hydroponic boxes (40x30x11.3 cm Alliber, Muehlheim, Germany) containing complete tomato nutrient solution (Ca(NO3)2 1.25 mol m–3, KNO3 1.5 mol m–3, MgSO4 0.75 mol m–3, K2HPO4 0.83 mol m–3, FeEDTA 0.05 mol m–3, H3BO3 11.6 mmol m–3, MnSO4 2.4 mmol m–3, ZnSO4 0.2 mmol m–3, CuSO4 0.1 mmol m–3, NaMoO4 0.1 mmol m–3) (K Koehl, personal communication based on Baumeister and Ernst, 1978) under a 12/12 h light/dark regime at 22/20 °C and constant (50%) relative humidity. After 1 week, plants were grown in different nitrogen conditions (saturated [8 mM ], replete [4 mM ] and deficient [0.4 mM ]). To minimize the effect of the altered nitrogen content on osmotic potential when nitrate was reduced to 0.4 mM, the nutrient solution was augmented by an equimolar mixture of calcium sulphate and potassium sulphate to maintain the same cation concentration as that of the nutrient medium. Similar adjustments were also made to balance the total nutrient content of the saturated nutrient solution with the other two nitrate-containing media. Plants were grown in two different light conditions, high light (900 µmol photons m–2 s–1) and in low light conditions (200 µmol photons m–2 s–1). Samples were taken in the exponential phase of grown with leaves harvested after 1, 24, 48, and 96 h of different nitrogen conditions or after 7, 14, and 21 d of different nitrogen conditions.
Chemicals
All chemicals were purchased from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany) with the exception of N-methyl-N-[trimethylsilyl]trifluoroacetamide (Macherey-Nagel GmbH & Co. KG, Düren, Germany).
Extraction, derivatization and analysis of tomato leaf metabolites using GC-MS
Metabolite analysis by GC-MS was carried out by a method modified from that described by Roessner et al. (2001). Tomato leaf tissue (100 mg) was homogenized using a ball mill precooled with liquid nitrogen and extracted in 1400 µl of methanol, 60 µl of internal standard (0.2 mg ribitol ml–1 water) was subsequently added as a quantification standard. The mixture was extracted for 15 min at 70 °C and mixed vigorously with 1 vol. of water. In order to separate polar and non-polar metabolites, 750 µl chloroform was then added to the mixtures. After centrifugation at 2200 g the upper methanol/water phase was taken and reduced to dryness in vacuo. Residues following reduction were redissolved in 40 µl of 20 mg ml–1 methoxyamine hydrochloride in pyridine and derivatized for 90 min at 37 °C followed by a 30 min treatment with 60 µl MSTFA (N-methyl-N-[trimethylsilyl]trifluoroacetamide) at 37 °C. 8 µl of a retention time standard mixture (0.029% (v/v) n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane, n-dotricontane, and n-hexatriacontane dissolved in pyridine) was added prior to trimethylsilylation. Sample volumes of 1 µl were then injected onto the GC column using a hot needle technique.
The GC-MS system used comprised an AS 2000 autosampler, a GC 8000 gas chromatograph and a Voyager quadrupole mass spectrometer (ThermoFinnigan, Manchester, UK). The mass spectrometer was tuned according to the manufacturer's recommendations using tris-(perfluorobutyl)-amine (CF43). Gas chromatography was performed on a 30 m Rtx_5Sil MS column with 0.25 µm film thickness and a 10 m Integra precolumn (Restek, Bad Homburg, Germany). The injection temperature was set at 230 °C, the interface at 250 °C and the ion source adjusted to 200 °C. Helium was used as the carrier gas at a flow rate of 1 ml min–1. The analysis was performed under the following temperature programme: 5 min isothermal heating at 70 °C, followed by a 5 °C min–1 oven temperature ramp to 350 °C and a final 5 min heating at 330 °C. The system was then temperature equilibrated for 1 min at 70 °C prior to injection of the next sample. Mass spectra were recorded at 2 scan s–1 with an m/z 50–600 scanning range. Both chromatograms and mass spectra were evaluated using the MASSLAB program (ThermoQuest, Manchester, UK) and the resulting data are prepared and presented as described in Roessner et al. (2001). The absolute concentrations of most metabolites were determined by comparison with calibration standard curve response ratios of various concentrations of standard substance solutions, including the internal standard ribitol and which were derivatized concomitantly with tissue samples.
Statistical analysis
If two observations are described in the text as different this means that their difference was determined to be statistically significant (P <0.05) by the performance of Student's t-tests.
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Results |
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Plant growth and nitrogen content
The aim of the work was to investigate the influence of different nitrogen supply on metabolism in tomato leaves. Wild-type tomato plants were grown in nitrogen-controlled conditions supplied either with a media saturated (8 mM
), optimal (4 mM ) or deficient (0.4 mM ) in nitrate concentration (K Koehl, personal communication based on Baumeister and Ernst, 1978). These experiments were performed in a high light intensity growth chamber (900 µmol photons m–2 s–1). After 7, 14, and 21 d under a given nutrient regime, metabolite profiling was used to evaluate the response of leaf metabolism to nitrate supply. In a parallel experiment the response of plants to the same nitrate treatments was performed under low light intensity (200 µmol photons m–2 s–1). A further, independent, experiment was performed to assess the short-term effects of nitrate nutrition on metabolite pools of tomato leaves. In this experiment, the metabolite profiles of tomato leaves at 1, 24, 48, and 96 h after alteration in nitrate supply were analysed in order to evaluate rapid metabolic responses to nitrate stress.
Gas chromatography-mass spectrometric (GC-MS) determination of metabolite levels
Having harvested leaf material from plants growing under different nutrient regimes, it was decided to characterize the biochemical phenotype they display. For this purpose, a GC-MS method was used for the analysis of tomato leaf metabolites that had previously been developed and thoroughly tested (Roessner-Tunali et al., 2003). An average of over 60 metabolites was detected across the nutrient regimes measured. Only the time zero control is presented in this paper, however, the full experimental data set can be viewed at JXB online. Although some of the changes in metabolite levels such as the variation in certain amino and organic acid contents are similar to those that have been documented previously in tobacco (Scheible et al., 1997), many of them are completely novel. In the subsequent sections the responses of certain compound classes to the various nutrient regimes applied are detailed.
Response of amino acid levels to altered nitrate supply
In plants growing under high light intensity the majority of the amino acids were observed to decrease progressively during the time-course of nitrogen starvation (Fig. 1A). Aspartate rapidly declined and was below the detection limit after 7 d of nitrogen starvation and for the remainder of the experiment. Alanine decreased progressively to less than half of the level found at the start of the experiment whilst arginine, glutamate, and tyrosine decreased to a quarter of the level found at the start of the experiment. The pattern of change in the glutamine and tryptophan pools was somewhat different, with an initial dramatic increase in levels followed by a subsequent rapid decline to less than 5% of its initial level. The level of tryptophan after 7 d treatment was remarkably high. By contrast, lysine and leucine increased marginally throughout the time-course of the experiment. Little variation was, however, observed in the metabolic pools of GABA, glycine, and valine.
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In parallel, tomato plants were also grown under nitrogen deficiency at a lower light intensity (Fig. 1B). Although far fewer amino acids could be detected, some of the trends described above were conserved under these light conditions with decreases observed in the levels of alanine, arginine, aspartate, and glutamate, although this was to a much lower extent than observed in high light. However, several differences in response between the two experiments are also apparent. In contrast to the plants growing in high light condition, increases in serine, ß-alanine and GABA (significant in the case of serine) were observed at the end of this experiment, whilst asparagine and threonine showed little change throughout the course of the experiment.
As a complementary approach, tomato wild-type plants were grown in nitrogen-saturated media under high-light conditions. Under these conditions a general trend of increased amino acids content is apparent (Fig. 1C). Arginine, GABA, lysine, phenylalanine, and tryptophan all show a biphasic response to nitrogen saturation, marginally declining initially before accumulating to levels between 2- and 15-fold of those observed at the start of the experiment.
A similar yet less pronounced tendency was also observed for alanine, glutamate, glycine, and methionine. The level of ornithine progressively increases during the experiment with aspartate, ß-alanine, glutamine, isoleucine, methionine, and valine following similar trends.
The relative level of cysteine and homoserine showed fluctuation during this experiment and, given that the nitrate-replete control for these three metabolites also fluctuated substantially throughout this time-course (see http://www.mpimp-golm.mpg.de/fernie for details), caution should be employed when interpreting these data. Plants growing in low-light conditions showed a similar pattern of change in metabolic poolsize following prolonged nitrogen saturation (Fig. 1D). GABA and tyrosine levels were significantly elevated at the end of the experimental period whereas glutamate and leucine also showed similar patterns of change to those observed previously. In general, only minor differences were observed in the pattern of response to nitrate saturation under the two different light regimes. However, as would be expected, the relative level of change is much reduced in the low-light-grown plants. A fact that is also reflected in the relatively low number of amino acids detected in leaf material harvested from these plants. When the results from these four treatments are compared they are largely fairly consistent. However, a few notable exception to this trend are apparent, for example, the levels of ß-alanine are high both in long-term nitrate starvation at low light and in long-term nitrate saturation at high light. In addition, the levels of lysine, homoserine, and leucine are anomalously high during nitrogen saturation at high-light intensity (lysine, homoserine) and low-light intensity (leucine), respectively.
In a separate experiment, metabolite levels were measured in plants exposed to altered nutrient regimes for only a short time (Fig. 1E, F). Over short periods of nitrogen deficiency, no clear tendency in amino acid composition was observed (Fig. 1E). The level of cysteine decreased significantly after only 1 h of nitrate deficiency. Increases in ß-alanine, glycine, isoleucine, phenylalanine and valine were also observed during nutrition starvation. Intriguingly, ß-alanine, isoleucine, and phenylalanine also increased significantly after short-term nitrate saturation under low-light intensity (Fig. 1F). Conversely, the level of GABA and lysine decreased significantly 1 h after changing to nitrogen-saturated media (but recovered to control levels thereafter). When these data are compared, it becomes clear that arginine, asparagine, and glutamate are surprisingly higher under nitrogen-deficient than nitrogen-saturated conditions.
Response of organic acid levels to altered nitrate supply
Growing wild-type plants in nitrate-deficient media under a high-light regime generally caused a dramatic reduction in the levels of major organic acids of the tricarboxylic acid cycle over time (Fig. 2A). Levels of citrate, isocitrate, fumarate, and malate demonstrated a rapid and significant reduction following the imposition of nitrate stress and, furthermore, remained low for the remainder of the experiment. A similar tendency was noted in succinate and 2-oxoglutarate pools, however, these pool sizes increased marginally over time following the dramatic decrease observed after 7 d of treatment. The levels of non-tricarboxylic acid cycle organic acids generally showed similar, if somewhat slower, responses to the stress imposed, with caffeate decreasing markedly at late stages of nitrate stress. Conversely, a dramatic and rapid increase was observed in the level of different forms of ascorbate and chlorogenate whilst a late increase in the level of glycerate was also observed following the imposition of nitrate stress.
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As observed for the amino acids, the pattern of response of organic acids to nitrate stress was largely conserved across the two light intensities used (compare Fig. 2A and B). The levels of citramalate, citrate, fumarate, isocitrate, malate, and succinate decreased sharply over the time-course of the experiment. In contrast, however, the levels of caffeate and shikimate tended, if anything, to decrease over the time-course of the experiment. Furthermore, whilst similar trends were observable in the levels of the various forms of ascorbate, the magnitude of these changes was greatly diminished under the low light regime. Similarly, chlorogenate was below the level of detection in samples harvested from these plants. The level of phosphate strongly fluctuated, with significant reduction in its levels being observed after 14 d but rising subsequently.
Following plant growth in nitrate-saturated media under high-light conditions, the levels of organic acids tended to increase (Fig. 2C), however, caution must be taken in interpreting these data as there is a large degree of fluctuation in the levels of several of these metabolites. Only aconitate and dehydroascorbate showed a significant decrease (after 7 d and 14 d of nitrogen saturation, respectively), whilst 2-oxoglutarate levels tended to increase with increased treatment time. The relative level of other forms of ascorbate, however, fluctuate somewhat during the time-course of the experiment. The levels of glucoronate, 6-P-gluconate, phosphate, and quinate all tended to rise during early stages of nitrogen saturation.
More spectacular changes were, however, observed in metabolite levels of nitrate-saturated-grown plants under low-light intensity (Fig. 2D) with aconitate, citramalate, isocitrate, malate, and phosphate all accumulating during the saturation period and caffeate increasing in the early stages of nitrate saturation and subsequently declining.
During short-term nitrogen starvation a significant reduction in the succinate pool was observed, as were fluctuations in the level of ascorbate (Fig. 2E). However, with the exception of these changes the organic acids pools largely remained constant. Similarly, there were few changes in relative metabolite level in plants grown in parallel but on nitrogen-saturated media (Fig. 2F). These were largely restricted to the increased level of ascorbate following saturation and fluctuations in the levels of succinate.
Response of carbohydrate levels to altered nitrate supply
In nitrogen-starved plants grown under a high-light regime there was a tendency for the levels of glucose-6-phosphate, maltose, and glycerol to increase with increased treatment time (Fig. 3A). An accumulation of inositol, maltitol, fructose 6-phosphate, and trehalose can also be observed, but only following prolonged nitrogen starvation. Maltose and xylose clearly decrease throughout the course of nitrate starvation. The levels of fructose, galactose, raffinose, and sucrose show no clear trend with respect to nitrate nutrition under these conditions. Conversely, nitrogen-deficient plants grown under low-light conditions display a clear trend to increasing carbohydrate content (Fig. 3B), with levels of arabinose, fucose, inositol, sucrose, and trehalose significantly increasing during the starvation period and mannose displaying a similar trend. Fructose, glucose, and maltose also accumulate after 7 d of growth on altered nutrient. However, it is worth noting that their levels diminish subsequently.
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Despite the relative constancy of carbohydrate pools in plants growing on saturated nitrogen media under high light intensity the levels of arabinose, maltitol, mannitol, and trehalose fluctuate across the time-course of the experiment (Fig. 3C). Carbohydrate levels of plants grown in parallel, but under low light intensity also remain relatively constant, irrespective of nitrate supply (Fig. 3D) with the increase in xylose at the onset of nitrogen saturation being the only obvious change.
The carbohydrate level was also assessed in plants growing in limited nitrogen for only a short period of time in order to gauge the short-term response to nitrate starvation (Fig. 3E). A significant increase in the level of arabinose was observed after 96 h of nitrogen starvation. Some fluctuation was also observed in the levels of inositol, raffinose, and trehalose, however, no clear trend was apparent in the pattern of change of these metabolites. Similarly, metabolite levels in plants growing in the same light conditions but on nitrogen saturation media did not show dramatic changes, with fluctuations in metabolite levels being confined to arabinose, glucose, and glycerol and minor increases observed in the level of maltose with increasing time in nitrate-saturated media.
Response of miscellaneous metabolite levels to altered nitrate supply
The levels of a range of other metabolites were also profiled in this experiment including fatty acids 16:0 and 18:0, dopamine, galacturonate, gluconate, glycerol 1-phosphate, nicotinate, norvaline, tyramine, and uracil. During long-term nitrate stress under a high-light regime the levels of the majority of these compounds were relatively stable, with the exception of the level of dopamine which significantly decreased after mid-term nitrogen deficiency and significantly increased after progressive nitrogen deficiency, and nicotinate which decreases progressively to levels below the level of detection at the end of the experiment with gluconate and norvaline following similar, if less extreme, trends (Fig. 4A). Perhaps surprisingly, no changes were observed in the level of ononitol throughout the time-course of the experiment. Following nitrogen starvation under a low-light regime, far fewer of these metabolites could be detected. However, of those that could, few of these varied significantly either (Fig. 4B). The exceptions to this being a similar yet less extreme decrease in the level of dopamine and a significant increase in uracil content at the end of the experimental treatment.
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When the plants were grown in nitrate-saturated conditions under a high-light regime there were also few changes in the levels of these metabolites. However, intriguingly, several of the trends in metabolite levels were the exact opposite of those described above for the nitrate-deficiency experiment, with increases being observed in the levels of nicotinate, noradrenaline and norvaline throughout the time-course of the experiment. Interestingly, the levels of ononitol increased following nitrate saturation in these conditions and there was a significant increase in the level of spermidine at the end of the experimental time-course (Fig. 4C). In the parallel experiment run under a low-light regime most of these metabolites were not detected, however, there was a minor increase in ononitol following nitrogen saturation and the levels of fatty acids 16:0 and 18:0 as well as glycerol 1-phosphate were significantly elevated following 14 d of high-nitrate treatment (Fig. 4D).
In the short-term nitrogen-deficiency experiment under the high-light regime very few changes in the levels of these metabolites were observed, with the exception that dopamine levels oscillated substantially and there was a significant decrease to levels below the limit of detection in nicotinate during the time-course of the experiment (Fig. 4E). In the short-term nitrogen-saturation experiment under the high-light regime no consistent pattern of metabolite change was observed (Fig. 4F).
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Discussion |
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Growth of tobacco plants on limited nitrogen supply has previously been demonstrated to lead to large decreases in 2-oxoglutarate, isocitrate, and malate, but to have little effect on 3-phosphoglycerate or phosphoenolpyruvate levels (Scheible et al., 1997
). Starch has also been demonstrated to accumulate in a wide range of species under conditions of restricted nitrogen nutrition (Rufty et al., 1988; Chu et al., 1992), this effect most probably being mediated through an up-regulation of AGPS transcription (Scheible et al., 1997). The response of the major soluble sugars to nitrate starvation is, however, less clear with both decreases (Scheible et al., 1997) and increases (Hofstra et al., 1985; Foyer et al., 1998) of these compounds being reported following nitrate stress in tobacco or supression of nitrate reductase activity in maize, respectively. For the purposes of the current study a GC-MS-based metabolic profiling approach was used to study a broader metabolic response to nitrogen nutrition under two different light intensities. This method, therefore, provides a large amount of data concerning changing metabolite patterns following nutritional treatment, but is by nature confined to small molecule metabolites (Fernie, 2003). Whilst the response of the primary metabolites to the imposed stresses can easily be rationalized, that of the secondary metabolites is less easy to interpret. That said, these data allow a comprehensive description of metabolic events following nitrate stress and highlight areas of interest for future study.
The importance of 2-oxoglutarate as a key regulator of carbon and nitrogen interactions has been noted in many previous studies (for a review see Stitt and Fernie, 2003). Interestingly, in the present study, the changes in the level of 2-oxoglutarate following nitrate deficiency were mirrored in the levels of other TCA cycle intermediates, suggesting a co-regulation of the cycle. Whilst the operation of the entire TCA cycle in the light remains somewhat contentious, more and more evidence is accumulating in support of the functioning of most of the enzymes of the cycle (Fernie et al., 2004b). Further support for the idea of co-regulation of this pathway exists in the literature with the transcript levels of the constituent enzymes responding in a synchronized manner (Lancien et al., 1999; Carrari et al., 2003b; Urbanczyk-Wochniak et al., unpublished data). Despite this fact, the genes encoding enzymes of the TCA cycle were not identified as nitrate regulated in the work of Crawford and co-workers (Wang et al., 2000, 2003). That said, there is a precedent for increases in the transcript levels of these genes since the nia mutant of tobacco was characterized by elevated transcripts of both citrate synthase and isocitrate dehydrogenase (Scheible et al., 1997).
In keeping with previous suggestions that 2-oxoglutarate and glutamate are key regulators of amino acid biosynthesis (Masclaux-Daubresse et al., 2002), these compounds increased in concert with a wide range of amino acids following transfer to nitrogen-saturated media. However, intriguingly in the current study, the fall in the level of several amino acids following transfer to nitrate-deficient media preceded a decrease in the level of glutamate (but not in the level of 2-oxoglutarate). As would be expected, amino acid levels generally decreased under nitrogen deficiency and conversely increased under conditions of nitrogen saturation. This was true across the range of amino acids with the exceptions of lysine and leucine which increased under both conditions (dependent on the light regime the plants were grown under) and GABA which was unaltered under nitrate deficiency in high-light conditions, but increased under nitrate deficiency in low-light conditions and under conditions of nitrate saturation. The level of GABA is clearly highly variant across the range of experimental conditions that have been applied. It has previously been suggested that the level of this amino acid may regulate the response of the highly interactive N assimilation, associated C metabolism, and photorespiratiory pathways to diverse environmental inputs (Foyer et al., 2003). Moreover, in analogy with mammalian systems a signalling role for GABA has recently been proposed in plants (Bouché and Fromm, 2004). The co-ordinated regulation of minor amino acids that was observed here has previously been commented on by Noctor et al. (2002) who suggested that factors early in the processes of C and N assimilation influence the relative levels of these metabolites. However, the results from the present study are difficult to interpret in this context and far more work, probably in a variety of species, is required to elucidate the mechanism and physiological importance of such signalling in plants. Particularly surprising in the data set presented here is the large accumulation of the amino acids that have a high synthetic cost, especially given the known feedback inhibition mechanisms of the enzymes involved in their synthesis, however, this too requires further experimentation before it can be fully understood.
The contents of a wide range of sugars also changed markedly with respect to nitrate supply, however, as would probably be expected, these changes were largely confined to nitrate deficiency with very few changes in the levels of these metabolites following nitrate saturation. The levels of many carbohydrates and associated metabolites increased following nitrate starvation with increases in the level of hexose phosphates, arabinose, fucose, and inositol as well as in maltitol and trehalose. These changes are, however, somewhat contrary to changes observed in transcript levels of Arabidopsis plants subjected to nitrate deficiency (Wang et al., 2003). This study revealed that, in addition to the known nitrate-regulated genes, several transcripts of primary metabolism are up-regulated following the resupply of nitrate following a period of stress including genes involved in glycolysis and trehalose synthesis. However, it must be noted that transcript levels do not necessarily directly correspond to the in vivo activity of the enzymes which they encode. By contrast with the general trend of increasing carbohydrate, levels of maltose and xylose decreased following nitrate stress. Since the importance of the hydrolytic pathway of starch breakdown has recently been demonstrated (Niittyla et al., 2004; Weise et al., 2004), and the accumulation of starch under nitrate stress is well-established, it is tempting to speculate that this minor decrease in maltose is the result of a reduced starch turnover.
Interestingly, the levels of non-TCA-cycle organic acids showed similar yet slower responses to the stresses imposed as the TCA cycle acids. The levels of caffeate decreased, whereas perhaps surprisingly the levels of ascorbate and chlorogenate increased following nitrate stress. By contrast, under nitrate saturation several of the minor organic acids increased including those such as ononitol which were unchanged under conditions of nitrate stress. These data are largely consistent with the studies of Stitt and co-workers who have recently performed detailed studies on the effect that modifying primary metabolism has on secondary metabolism (Henkes et al., 2001; Matt et al., 2002). However, it should be noted that previous studies of transgenic tobacco plants exhibiting decreased Rubisco expression revealed that these plants were characterized by an inhibition of nitrate reductase and a decrease in the level of chlorogenate (Matt et al., 2002). Since study of the interaction between primary and secondary metabolism is in its infancy, it is, however, clear that further experimentation is required to clarify the biological relevance of these observations.
In conclusion, the data presented here suggest that nitrate nutrition has wide-ranging effects on plant leaf metabolism with nitrate deficiency resulting in decreases in many amino and organic acids and increases in the level of several carbohydrates and phosphoesters, as well as a handful of secondary metabolites. The results of these studies confirm and extend previous studies of nutrient deficiency in tobacco (Matt et al., 2001a, 2002; Masclaux-Daubresse et al., 2002). Conversely, growth in nitrate-saturated media largely resulted in opposing changes in these metabolite levels. However, in some instances, such as the carbohydrate levels and the secondary metabolites, this was not the case. Interestingly, these changes were largely conserved across the two different light intensities with the exception that changes observed at the lower light intensity were generally less marked. In addition, the changes in carbohydrate levels were generally larger between the different light conditions than they were between the different nutrient regimes. The short-term responses of metabolite levels to perturbation of nitrate nutrition were, however, far less predictable, most probably as the plant needs time to adjust to its conditions and the metabolite level is somewhat downstream of the initial responses of altered gene expression, protein stability etc. It is, therefore, clear that more studies are required to understand fully the complex regulation involved in the metabolic networks affected. Future studies will build on this study of the Aco1 mutant of Solanum pennellii (Carrari et al., 2003a) by focusing on the evaluation of the relative roles of aconitase, citrate synthase, and the various isoforms of isocitrate dehydrogenase in amino acid biosynthesis and related processes.
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Supplementary data |
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Seven tables containing the full experimental data can be found at JXB online.
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Acknowledgements |
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We thank Dr Karin Koehl for great help in establishing the tomato hydroponic conditions. The authors acknowledge the financial support of the Max Planck Society.
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References |
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