Journal of Experimental Botany, Vol. 53, No. 370, pp. 959-970,
April 15, 2002
© 2002 Oxford University Press
Original Papers |
Steps towards an integrated view of nitrogen metabolism
1 Max-Planck-Institute for Molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany
2 Dipartimento di Scienze della Vita, Seconda Universita di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
3 Instituto de Recursos Naturales y Agrobiologia de Salamanca, CSIC, 37008 Salamanca, Spain
Received 18 July 2001; Accepted 16 October 2001
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Abstract |
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 Top Abstract IntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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This article discusses how nitrate assimilation is integrated with nitrate uptake, with ammonium assimilation and amino acid synthesis, with pH regulation, and with the sugar supply in tobacco leaves. During the first part of the light period, nitrate assimilation exceeds nitrate uptake by 2-fold and ammonium assimilation by 50%, leading to rapid depletion of nitrate and accumulation of ammonium, glutamine, glycine and serine. NIA, NII and PPC expression show a shared maximum early in the diurnal cycle to direct carbon towards malate synthesis for pH regulation. Later in the diurnal cycle an orchestrated increase of GLN2, PKc, CS, and ICDH-1 expression re-establishes a balance between nitrate assimilation and ammonium metabolism. Nitrate uptake continues throughout the night, replenishing the leaf nitrate pool. These diurnal changes are attenuated or abolished in mutants with low NIA activity, and modified in wild-type plants growing on different nitrogen sources or elevated [CO2]. Comparison across genotypes and conditions reveals that NIA transcript levels are always closely related to the balance between nitrate influx and assimilation, but are unrelated to changes of glutamine or 2-oxoglutarate. In a systematic search for other downstream regulators, a wide range of downstream metabolites was fed to detached leaves and glutamate, cysteine, asparagine, and malate identified as candidates. Low sugars totally inhibit nitrate assimilation, overriding signals from nitrogen metabolism. Moderate changes act post-transcriptionally, and larger changes lead to a collapse of the NIA transcript. Low sugars also lead to a collapse of minor amino acids and a dramatic decrease of phenylpropanoids and nicotine. Consequently, wild-type plants growing in unfavourable light regimes and antisense RBCS transformants are simultaneously carbon- and nitrogen-limited.
Key words: Amino acids, GOGAT, malate, NIA, nitrate reduction, nitrogen metabolism, pH.
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Introduction |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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At first sight, nitrate is utilized in a linear pathway that involves the uptake and transport of nitrate within the plant, followed by nitrate assimilation, ammonium assimilation, amino acid biosynthesis, and protein synthesis. There are, however, complex interactions with many other aspects of nitrogen metabolism, including (i) the storage and remobilization of nitrate in different parts of the plant, (ii) de novo ammonium assimilation, (iii) the recycling of ammonium released during photorespiration (Hirel and Lea, 2001
), (iv) the distribution of nitrogen between the highly branched pathways of amino acid biosynthesis (Morot-Gaudry et al., 2001), and (v) the multifarious fates of amino acids, which can be exported, stored in the vacuole, used for protein synthesis, or diverted into secondary metabolic pathways leading to phenylpropanoids, alkaloids and tetrapyrroles (Heldt, 1996). There is also a complex interaction with carbon metabolism which provides (vi) malate as a counter-anion to prevent alkalinization (Martinioa and Rentsch, 1994), (vii) 2-oxoglutarate as the primary acceptor of ammonium in the GOGAT pathway (Heldt, 1996) and (viii) numerous other organic acids and phosphorylated intermediates that are required as carbon precursors in the various amino acid pathways (Morot-Gaudry et al., 2001). Further, (ix) reactions in photosynthesis or carbohydrate breakdown are required to generate the reducing equivalents that are consumed during the reduction of nitrate to ammonium (Kaiser et al., 2000; Foyer et al., 2001).
Nitrogen, therefore, moves along a complex branching and merging pathway which interacts at numerous sites with the carbon flow, pH regulation, and ion and assimilate flow at the cell and whole plant level. Superimposed on these metabolic fluxes is the far-reaching influence of nitrate and nitrogen metabolism on plant development and architecture, including changes in root architecture, the timing of senescence, and flowering (Stitt, 1999; Stitt and Krapp, 1999; Brouquisse et al., 2001; I Loef, M Stitt, unpublished data). One of the challenges of research in nitrogen metabolism is to develop methodologies and approaches to analyse this network. In other reviews, the role of nitrate and nitrogen metabolites in regulating metabolism and development has been discussed (Stitt, 1999; Stitt and Scheible, 1999; Stitt and Krapp, 1999; Crawford 1995). The aim of this review is to summarize recent work of investigations into the ways that nitrate uptake, nitrate assimilation and ammonium assimilation interact with each other and with events further downstream in nitrogen metabolism and carbon metabolism in source leaves of tobacco.
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Nitrogen metabolism undergoes dramatic diurnal changes, which are driven by a transient imbalance between the rate of nitrogen assimilation and the rate of nitrate uptake and ammonium assimilation in the first part of the light period |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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Nitrate assimilation in tobacco leaves is characterized by dramatic diurnal changes in gene expression, enzyme activities, metabolite levels and fluxes. These provide an ideal system to illustrate and analyse the interactions between nitrate uptake, nitrate assimilation, and downstream events in nitrogen and carbon metabolism.
When tobacco plants are growing in high nitrate and a favourable light regime, their leaves contain high levels of the NIA transcript at the end of the night. Illumination stimulates translation of the transcript and inhibits degradation of NIA protein (Kaiser et al., 1999), leading to an approximately 3-fold increase of NIA protein during the first hours of the light period (Scheible et al., 1997b). Illumination also leads to rapid post-translational activation of NIA (Kaiser et al., 1999). As a result, high rates of nitrate assimilation are achieved during the first part of the light period. These exceed the rate of nitrate uptake by a factor of two (Matt et al., 2001a), leading to a rapid depletion of the leaf nitrate pool. The rate of nitrate assimilation also exceeds net flux though the GOGAT pathway by about 25%, leading to accumulation of reduced nitrogen in immediate downstream products like ammonium and glutamine, as well as in the photorespiratory metabolites glycine and serine (Scheible et al., 2000; Matt et al., 2001a).
This imbalance between the rate of nitrate reduction and the events lying upstream and downstream from NIA is corrected later in the diurnal cycle. Nitrate assimilation is progressively inhibited by mechanisms that act at several levels to decrease NIA activity. These include a dramatic decrease of the NIA transcript level which commences soon after illumination (Scheible et al., 1997b, 2000; Geiger et al., 1998; Matt et al., 2001a) and results in a decline of NIA protein and activity during the second part of the light period (Scheible et al., 1997b) as well as post-translational inactivation of NIA after darkening (Kaiser and Huber, 1994; Scheible et al., 1997b; MacKintosh, 1998). As a result, the rate of nitrate reduction falls about 2-fold in the second part of the light period, and is negligible during the night (Matt et al., 2001a). Changes of the cytosolic NADH concentration might also affect in vivo NIA activity (Kaiser et al., 2000).
By contrast, nitrate uptake in the roots and throughput of nitrate to the shoot remains high during the entire light period and falls by only 30% during the night (Matt et al., 2001a). The nitrate that is taken up during the night is used almost exclusively 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 during the night (Matt et al., 2001a). The reciprocal regulation of NIA and glutamine synthetase facilitates the remobilization of accumulated downstream products later in the light period and during the night. When tobacco is grown on ammonium nitrate, in which conditions ammonium accumulates to much higher levels during the day and remains high for most of the night, glutamine synthetase activity remains high throughout the night (Matt et al., 2001b).
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Excess NIA capacity allows rapid changes of nitrate assimilation in response to environmental conditions, and also buffers the rate of nitrate assimilation against long-term changes |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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Wild-type tobacco plants growing in a favourable light regime and with a high nitrate supply therefore possess excess capacity for nitrate assimilation, which is progressively inhibited as the diurnal cycle proceeds. Independent evidence that wild-type tobacco possesses excess capacity for nitrate reduction was provided by earlier studies of mutants with decreased NIA activity, in which it was demonstrated that tobacco mutants with one or two instead of four functional NIA gene copies grow as fast as wild-type tobacco and contain comparable levels of protein and chlorophyll (Scheible et al., 1997b
). A similar picture has been reported for mutants with decreased NIA expression in a range of other species including N. plumbiginifolia, Arabidopsis and barley (see Stitt and Krapp, 1999, for references).
The interpretation of the diurnal changes developed earlier (Matt et al., 2001a, b) clarifies how the mutants compensate for their lower NIA activity. In wild-type plants, maximum rates of nitrate assimilation are realized for a short time during the first part of the light period and are then inhibited, because they are in excess of the requirements in the rest of the pathway. In low-NIA mutants, where the rate of nitrate assimilation is lower during the first part of the diurnal cycle, this imbalance does not develop and the feedback inhibition later in the diurnal cycle is attenuated or abolished. Thus, NIA protein and NIA activity are 2–3-fold lower than in wild-type plants during the first part of the light period, but the decline of NIA protein and activity later in the light period is abolished and the post-translational inactivation of NIA during the night is partially reversed (Scheible et al., 1997b). This allows a higher rate of nitrate assimilation later in the diurnal cycle and compensates for the lower rate in the first part of the light period.
The excess NIA capacity in wild-type tobacco allows nitrate reduction to be rapidly stimulated when conditions change. For example, in experiments in which wild-type tobacco and F23xF22, F22xNia30 and F23xNia30 genotypes (containing 4, 3, 1, and 1 functional NIA genes, respectively) were grown for several weeks on very low nitrate and then fertilized with nitrate, or were grown for several weeks under low light and the light intensity then increased, the rate of increase of NIA activity following the transition was directly proportional to the number of functional NIA genes (N Bujard, A Krapp, M Stitt, unpublished data). Excess NIA capacity might also allow a higher rate of nitrate assimilation to be maintained in unfavourable conditions than would otherwise be possible, or in conditions where high rates of nitrate reduction can only be achieved for a short period in each diurnal cycle.
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Nitrate leads to an orchestrated change in gene expression, which facilitates a reprogramming of carbon metabolism |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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Nitrate assimilation is closely integrated with changes in organic carbon metabolism. During nitrate assimilation, carbohydrate synthesis is decreased and more carbon is converted via glycolysis to phosphoenolpyruvate and enters organic acid metabolism. Organic acid metabolism has two distinct functions during nitrate assimilation. (i) Phosphoenolpyruvate carboxylase (PPC) operates together with malate dehydrogenase to provide malate, which acts as a counter-anion and prevents alkalinization during nitrate assimilation (Martinoia and Rentsch, 1994
). Malate is exported to the roots, where it is decarboxylated. (ii) PPC operates together with pyruvate kinase (PK), the mitochondrial citrate synthase (CS), pyruvate dehydrogenase and the cytosolic NADP-dependent isocitrate dehydrogenase (NADP-ICDH) to provide 2-oxoglutarate, which is the primary carbon acceptor for ammonium.
The studies with Nia30(145) tobacco mutants which have very low NIA activity and therefore accumulate nitrate, but have very low levels of reduced nitrogen compounds, showed that nitrate induces several genes encoding enzymes involved in organic acid synthesis (Scheible et al., 1997a). These include PPC (encoding phosphoenolpyruvate carboxylase), PKc (encoding cytosolic PK), CS (encoding mitochondrial CS), and ICDH1 (encoding cytosolic NADP-ICDH). This reprogramming of gene expression results in an increase of PPC activity, accumulation of 2-oxoglutarate, malate, and other organic acids. Nitrate also represses AGPS (Scheible et al., 1997a), which encodes the regulatory subunit of ADP-glucose pyrophosphorylase (AGPase), a key enzyme for the regulation of starch synthesis. The stimulation of flux to organic acids leads to a decrease of phosphoenolpyruvate and 3-phosphoglycerate, resulting in allosteric inhibition of AGPase and a further inhibition of starch synthesis (Scheible et al., 1997a). These changes in transcript and metabolite levels also occur in F22 and F23 tobacco mutants, in which the decrease of NIA activity is much smaller and the increase of nitrate is in the range experienced in wild-type plants in normal conditions (Scheible et al., 2000). This clearly establishes the physiological relevance of these findings. Interestingly, no evidence was found in tobacco for an inhibition of sucrose synthesis. Continued synthesis of sucrose may be important to allow export of amino acids from the leaf. It is not known if a different response occurs in other species, which make less starch.
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The expression and activity of enzymes in nitrogen and carbon metabolism is regulated in a co-ordinate pattern, with two different groups of genes showing out-of-phase changes that are related to changing priorities in the allocation of carbon to the synthesis of malate and carbon acceptors for reduced nitrogen |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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Recent results (Scheible et al., 2000
) reveal that carbon metabolism is exquisitely co-ordinated with the diurnal changes of nitrate and ammonium metabolism. The levels of the transcripts for PPC, PK, CS, and ICDH1 show large diurnal changes in tobacco leaves. These are accompanied by changes of the activities of the encoded enzymes, indicating that the diurnal changes of transcript levels contribute to the changes of enzyme activity, although of course not excluding further contributions from post-transcriptional regulation and protein turnover (as are known to occur for NIA; Kaiser et al., 1999; Stitt and Krapp, 1999). The diurnal changes of these transcripts and enzyme activities are modified in nitrate-limited wild-type plants and are suppressed in genotypes with low NR activity (Scheible et al., 2000), demonstrating that they are driven by signals derived from nitrate and/or the metabolism of nitrate.
The genes encoding enzymes in nitrate, ammonium and organic acid metabolism can be divided into two blocks, which differ with respect to the timing of the diurnal change and the sensitivity with which their expression responds to changes in nitrogen metabolism. The first group, which includes NIA, NII and PPC, shows two main characteristics (Scheible et al., 1997b, 2000): (i) the transcript level is highest at the end of the night and falls rapidly during the light period and the encoded enzyme activity rises to a maximum during the first part of the light period and declines in the second part of the light period and (ii) the transcript level and activity of the encoded enzyme respond very strongly to changes in nitrate fertilization. The second group includes PKc, CS, ICDH1 (Scheible et al., 2000), and GLN2 (Matt et al., 2001a, b). For this group, transcript levels and encoded activities are highest at the end of the light period and the first part of the dark period. Although expression is affected by manipulation of nitrogen metabolism, the changes are less dramatic than for the first group of enzymes.
The reciprocal expression of these two groups of genes leads to a marked switch of metabolism. During the first part of the light period, when NIA and PPC activity are at a maximum and glutamine synthetase, PK, CS, and NADP-ICDH activity are low, there is an accumulation of malate, citrate decreases, and glutamine and ammonium accumulate. Later in the light period and during the dark period when NIA and PPC activity have decreased and glutamine synthetase, PK, CS and NADP-ICDH activity are at their diurnal maximum, malate decreases, citrate increases and ammonium and glutamine decrease. The amplitude of these diurnal changes of metabolite levels is large, compared to the total flux through these pools in a diurnal cycle, showing that the changes in expression has a marked impact on fluxes. The amount of malate that accumulates during the day is equivalent to about 30% of the nitrate assimilated per day (Scheible et al., 1997b, 2000). Assuming that one molecule of malate is synthesized per molecule of nitrate assimilated, this indicates that the rate of nitrate assimilation exceeds the rate of malate export by about 50%. The diurnal changes of citrate are equivalent to about 10% of the total carbon required during the conversion of nitrate to amino acids (Scheible et al., 2000). The accompanying increase of ammonium and glutamine allows 15–20% of the assimilated nitrate to be temporarily accumulated in a form that contains no, or relatively low, carbon (Matt et al., 2001a).
These results reveal that there is a shift in the priorities for carbon use during the diurnal cycle. During the first part of the light period, when nitrate assimilation is maximal, a co-ordinated increase of PPC and NIA expression prioritizes malate synthesis for pH regulation. The leaf has a low buffer capacity, making it essential to tightly co-ordinate processes that contribute to pH regulation with the current rate of nitrate assimilation. On the other hand, provision of carbon skeletons like 2-oxoglutarate may not have to be so tightly linked to nitrate assimilation, because a temporary imbalance can be buffered by depleting the citrate pool and by allowing reduced nitrogen to accumulate in intermediates like ammonium and glutamine. Later in the day when intermediates have accumulated in the pathway of ammonium assimilation and, perhaps even more seriously, in the photorespiratory pathway, nitrate assimilation decreases and more carbon is diverted towards the production of 2-oxoglutarate, in order to facilitate remobilization of ammonium and glutamine. The diurnal changes of expression of the enzymes in carbon metabolism are therefore part of a larger orchestrated programme that allows de novo nitrate and ammonium metabolism to be integrated with the regulation of cellular pH and the massive flows of ammonium associated with photorespiration, while allowing the burden on carbon metabolism to be spread across a wide part of the diurnal cycle.
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Nitrate and ammonium assimilation are regulated transcriptionally, in response to the balance between nitrate influx and nitrate assimilation, and post-translationally in response to downstream signals from N metabolism |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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The observation that the diurnal changes of NIA, NII, PPC, PKc, CS, and ICDH1 expression are attenuated or abolished in mutants with decreased NIA activity and are strongly modified in wild-type plants when the nitrate supply is low (Scheible et al., 2000
) shows that they are driven by events related to nitrogen metabolism. Nitrate reduction is regulated at several levels and in response to a variety of signals (see above: Kaiser et al., 1999; Meyer and Stitt, 2001). Briefly, nitrate induces NIA, sugars are thought to induce NIA, and glutamine or a related metabolite is thought to repress NIA (Hoff et al., 1994; Meyer and Stitt, 2001; Foyer et al., 2001). The position with respect to glutamine is rather controversial. Whereas early studies found a negative correlation between glutamine and the NIA transcript level during diurnal changes and showed that inhibition of glutamine synthetase activity by phosphinothricin leads to a decrease of glutamine and induction of NIA transcript (Deng et al., 1991), it was subsequently reported (Dzuibany et al., 1998) that a large decrease of GOGAT activity and accumulation of glutamine in a gluS mutant of Arabidopsis does not lead to a decrease of the NIA transcript level.
A large data set has been collected describing the diurnal changes of transcripts, protein levels or enzyme activities and overall metabolite levels in wild-type tobacco growing in ambient and elevated [CO2] (Geiger et al., 1998; Matt et al., 2001b), in nitrate and ammonium nitrate (Matt et al., 2001b), in high and low nitrate (Scheible et al., 1997b), and in mutants with a small and large decrease in NIA activity (Scheible et al., 1997b; Krapp et al., 1998; Scheible et al., 2000). Inspection of this data set leads to the following conclusions about the role of different signals in regulating NIA expression.
First, there is always a close relationship between the diurnal changes of leaf nitrate and the diurnal changes of the NIA transcript level. The crucial factor is not the absolute amount of nitrate in the leaf but, rather, the direction in which it is changing. Leaf nitrate and NIA transcript fall during the day and recover during the night. When recovery of leaf nitrate is slowed due to lower rates of nitrate uptake in the roots (e.g. in plants growing in elevated [CO2] on ammonium nitrate; Matt et al., 2001b) the recovery of the NIA transcript is also delayed and weakened. These results are interpreted as evidence that NIA transcript responds to the balance between the influx and the reduction of nitrate, which (as discussed already) changes dramatically during the diurnal cycle. Most of the nitrate in the leaf is located in the vacuole and the cytosolic nitrate concentration changes markedly between the dark and light in leaf cells, in contrast to root cells where it remains remarkably constant (AJ Miller, personal communication). The most likely candidate for the immediate signal is therefore a change of the cytosolic nitrate concentration, although more experiments are required to provide direct evidence for this proposal. The same signal might also initiate the dramatic changes in the flux of nitrate out of and into the vacuole that will be required during the day and night, respectively.
Second, there is no consistent relationship between the NIA transcript level and the level of glutamine. Although the results confirm that these parameters are negatively correlated during the diurnal changes in plants growing on nitrate, this relationship breaks down in many other conditions. In elevated [CO2], the decrease of the NIA transcript after illumination is just as rapid as in ambient [CO2], even though glutamine rises more slowly (Geiger et al., 1998; Matt et al., 2001b). When tobacco is grown on ammonium nitrate instead of nitrate, glutamine is much higher and the diurnal changes of glutamine are strongly damped, but the absolute level and the diurnal changes of the NIA transcript resemble those found in nitrate-grown plants (Matt et al., 2001b).
2-Oxoglutarate acts antagonistically to glutamine in bacteria and fungi (see Stitt and Krapp, 1999, for references). It has been suggested (Ferrario-Méry et al., 2001; Foyer et al., 2001) that parallel changes of 2-oxoglutarate may explain why the NIA transcript level is not always negatively correlated with glutamine. This does not explain the discrepancy between the changes of glutamine and the NIA transcript in our experiments. For example, when tobacco is grown with nitrate in elevated [CO2], glutamine is decreased and 2-oxoglutarate is unaltered, but the NIA transcript level resembles that in ambient [CO2]. When tobacco is grown with ammonium nitrate in elevated [CO2], glutamine is markedly decreased and 2-oxoglutarate is unaltered, but the NIA transcript level is lower than in ambient [CO2] (Matt et al., 2001b). Further, when 2-oxoglutarate is supplied via the petiole to detached leaves there are only small and inconsistent changes in the NIA transcript level and no changes of NIA activity, even though the internal pool of 2-oxoglutarate increases several fold (Müller et al., 2001). These results appear to contrast with those of Ferrario-Méry et al. who found that floating tobacco leaf discs on glutamine or 2-oxoglutarate led to a decrease and increase of the NIA transcript level, respectively (Ferrario-Méry et al., 2000). Further experiments are needed to understand these differences and the possibility cannot be excluded that NIA expression may be regulated by a small sub-pool of glutamine or 2-oxoglutarate, which changes independently of the overall leaf content. Nevertheless, these results indicate that these two metabolites do not always play a major role in the feedback regulation of NIA expression and open the question whether there are further feedback mechanisms that have been overlooked to date. In this context, two differences between plants and microorganisms should be noted. First, nitrate is the most important source of mineral nitrogen for plants, whereas ammonium is more important for microbes. Second, in the leaves of many plants glutamine may not always be a reliable parameter to monitor internal nitrogen status. The reassimilation of ammonium released during photorespiration in C3 plants accounts for up to 90% of the flux through the GOGAT pathway in leaves of C3 plants. When the rate of photorespiration changes, the levels of intermediates in this pathway including glutamine will be subject to large changes, for reasons that are extraneous to the net assimilation of nitrate and the nitrogen status of the plant.
Third, post-transcriptional mechanisms contribute to the regulation of nitrogen metabolism. Experiments by Kaiser and coworkers (see above) have shown that photosynthesis or related changes in sugars affect the synthesis and degradation of NIA protein (see above), and the results from our laboratory indicate that high glutamine leads to a decrease of NIA activity, which occurs independently of changes in the NIA transcript level. For example, in mutants with a reduced number of functional NIA gene copies (Scheible et al., 1997b), lower levels of glutamine are associated with a slower decrease of NIA protein and activity in the second part of the light period whereas NIA transcript levels fall in the same way as in wild-type plants. When tobacco is grown on ammonium nitrate instead of nitrate, NIA transcript levels are unaltered, but there is a decrease of NIA activity throughout the whole diurnal cycle, which correlates with higher levels of glutamine than in nitrate-grown plants (Matt et al., 2001b). The present results also indicate that ammonium or a related metabolite increases the activity of glutamine synthetase, again acting via a post-transcriptional mechanism (Matt et al., 2001b). The mechanisms involved still have to be characterized.
Finally, our results reveal that changes of sugars do not play a key role in the regulation of NIA expression in growth conditions where the light regime is favourable for photosynthesis. The NIA transcript level is highest at the end of the night, when leaf sugars are at a minimum, and growth in elevated [CO2] failed to lead to an increase of NIA expression. As will be discussed later, sugars do have a dramatic effect on NIA expression, but only when they fall to lower levels.
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Glutamate exerts feedback regulation on nitrate assimilation |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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As it was not possible to find any convincing evidence that glutamine or 2-oxoglutarate play a dominant role in the feedback regulation of NIA expression in the conditions used in these experiments, a systematic search for other metabolites that might exert feedback regulation on nitrate assimilation was recently initiated. Tobacco leaves were detached at the start of the light period and a wide range of possible candidates supplied via the petiole, including not only the immediate products of nitrate and ammonium assimilation, but also metabolites formed further downstream in nitrogen metabolism and in related aspects of metabolism and cell function, including photorespiration and pH regulation.
Ammonium and glutamine had no effect on NIA transcript levels, and led to no or only a small decrease of NIA activity, even though their internal pools increased dramatically (C Müller, M Stitt, unpublished data). This confirms the conclusions drawn from the diurnal changes of metabolites and transcript levels (see above).
Addition of glutamate, by contrast, led consistently to a marked decrease of the NIA transcript level and a 50% decrease of NIA activity down to the level typically found in the dark (C Müller, M Stitt, unpublished data). The decrease of NIA activity was due mainly to a decrease of Vmax activity, whereas the activation state decreased only slightly. Intriguingly, supplying glutamate led to only a small increase of the internal glutamate pool, indicating either (i) that NIA is responding extremely sensitively to the glutamate pool, or (ii) that the signal is involved in events related to the metabolism of glutamate.
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GOGAT and/or glutamate dehydrogenase represent an important site for the regulation of ammonium metabolism |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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Feeding glutamate led to an increase of ammonium and an even larger increase of glutamine. This observation indicates that glutamate plays a pivotal role in a sensitive feedback mechanism that regulates ammonium assimilation. This interpretation is supported by the diurnal changes of metabolite levels. Whereas glutamate is remarkably constant throughout the diurnal cycle and across a range of growth conditions (Geiger et al., 1998
; Scheible et al., 2000; Matt et al., 2001a) there are large diurnal changes of ammonium and glutamine, about 15–20% of the total reduced nitrogen moving through leaf metabolism each day (Matt et al., 2001a, b). The transient accumulation and remobilization of reduced nitrogen in intermediates immediately upstream from GOGAT indicates that the flow of reduced nitrogen into metabolism is regulated at this point. When 2-oxoglutarate was supplied to leaves there was a small decrease of glutamine and a large increase of glutamate (C Müller, M Stitt, unpublished results), providing evidence that the endogenous concentration of 2-oxoglutarate restricts in vivo GOGAT activity. This underlines the importance of regulation mechanisms that affect the formation of 2-oxoglutarate (see below). To investigate whether GOGAT itself is subject to allosteric or post-translational regulation, novel methods are being developed for the assay of GOGAT (Y Gibon, P Carillo, unpublished data). As discussed below, preliminary evidence indicates that GOGAT activity falls when sugars decrease. Expression of GLU, which encodes ferredoxin-dependent GOGAT, is stimulated by nitrate (Scheible et al., 1997a). Further studies are needed to investigate if expression is also affected by other parameters related to nitrate and nitrogen metabolism. This study's interpretation implies that plants differ from microbes, where the rate of ammonium assimilation is regulated at the initial step catalysed by glutamine synthetase. The amount of ammonium that can be accumulated in a plant tissue may be constrained because ammonium is an effective uncoupler of thylakoid electron transport, and because ammonia will be lost through the stomata. This provides a rationale to accumulate glutamine and to regulate the net rate of ammonium assimilation at the level of the reaction catalysed by GOGAT.
Intriguingly, after feeding glutamate there was also an increase of 2-oxoglutarate (C Müller, M Stitt, unpublished results). This is consistent with equilibration of glutamate and 2-oxoglutarate via aminotransferase reactions. An alternative, and not incompatible, explanation is that glutamate is converted to 2-oxoglutarate via glutamate dehydrogenase. This would provide a further explanation for the increase of ammonium after feeding glutamate. It can be speculated that glutamate dehydrogenase might readjust the relative levels of glutamate and 2-oxoglutarate when, for some reason, these move into a range that is inappropriate for efficient operation of GS, GOGAT or the various aminotransferases that utilize glutamate. The complex structure of the GOGAT pathway and the following steps, with repeated loops in which the product at one step is cycled back to act as a substrate at an earlier step, may make it necessary to regulate the concentrations of these rapidly turning-over pools carefully. A further possibility is that glutamate dehydrogenase forms a cycle with glutamine synthetase and GOGAT to increase the sensitivity with which the overall rate of ammonium assimilation responds to changes in the levels of ammonium, glutamate and 2-oxoglutarate.
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A systematic search for further feedback mechanisms indicates that specific minor acids including cysteine and asparagine exert feedback regulation on nitrate assimilation |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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In micro-organisms, the fluxes into the various pathways of amino acid biosynthesis are subject to an exquisite interacting feedback network, which maintains the levels of the individual amino acids in a range appropriate for reliable loading of tRNA and error-free protein synthesis. There is good evidence for a similar situation in plants (Lea and Forde, 1994
; Morot-Gaudry et al., 2001). The present study's experiments concentrated on investigating whether the levels of precursors in central nitrogen metabolism restricts the rate of amino acid synthesis, and whether any of the minor amino acids exert feedback control on the early steps in nitrate and ammonium metabolism. Addition of ammonium, glutamine, glutamate, and 2-oxoglutarate did not lead to a marked increase of the levels of the minor amino acids (C Müller, M Stitt, unpublished data), indicating that flux into the amino acid biosynthesis pathways is not limited by the availability of glutamate or other amino donors in central metabolism, at least in plants where the nitrogen supply is not limiting. Further studies are needed, however, to investigate whether the same holds in nitrogen-limited plants.
To investigate whether minor amino acids exert feedback regulation on nitrate or ammonium metabolism, a cocktail of Phe, Tyr, Trp, Leu, Ileu, Val, Thr, His, Arg, Asn, Met, and Cys was prepared with the individual amino acids in the same relative levels as found in a leaf, and supplied via the petiole to detached tobacco leaves. This cocktail led to a 2–3-fold decrease of NIA activity, when it was supplied in the presence of nitrate, nitrate plus ammonium, or nitrate plus glutamine, and also resulted in a small additional inhibition of NIA activity when it was supplied in the presence of nitrate plus glutamate (C Müller, M Stitt, unpublished data). This inhibition of NIA activity was not due to an increase of glutamate or other amino acids in central metabolism.
To elucidate whether this inhibition is due to a mechanism that integrates changes in the levels of all these minor amino acids or if one or a small number of specific members are involved, further experiments were carried out in which each minor amino acid was supplied individually. When added individually cysteine and, to a lesser extent, asparagine were inhibitory (C Müller, R Morcuende, M. Stitt, unpublished results). Further experiments are continuing to analyse the impact on NIA transcript levels. Nevertheless, when the cocktail was prepared omitting cysteine, asparagine and (see below) serine, it still led to a decrease of NIA activity indicating that further as yet unidentified combinations of minor amino acids are also effective.
The three amino acids identified to date as feedback inhibitors of nitrate reduction, glutamate, cysteine and asparagine, occupy strategic positions in amino acid metabolism. Glutamate represents the first product of nitrate and ammonium assimilation that is not subject to large extraneous changes during the diurnal cycle, and is also the immediate or indirect starting point for the synthesis of most amino acids and nitrogenous secondary metabolites in the plant. According to our interpretation of the results from feeding experiments, it is also immediately downstream of the main site(s) for regulation of ammonium assimilation. Glutamate is already known to be an important allosteric regulator of PEPC and pyruvate kinase (Turpin et al., 1997). Further, plants contain homologues to the glutamate receptor proteins found in animals (Hsieh et al., 1998). Cysteine represents the site at which sulphur enters amino acid metabolism. It is attractive to hypothesize that cysteine, or a related metabolite, regulates nitrogen assimilation in response to changes in sulphur metabolism. Asparagine is noteworthy because it responds in a diametrically different manner to other amino acids in response to carbohydrate depletion. Low sugars induce ASN1 and lead to an increase of asparagine (Lam et al., 1996), whereas all other minor amino acids (see below) decrease when sugars are low. Feedback inhibition by asparagine might, therefore, provide one mechanism to inhibit nitrate reduction when sugars are low.
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There is no evidence for a strong feedback inhibition by serine or glycine |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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De novo assimilation of nitrate and ammonium interacts closely with the recycling of ammonium during photorespiration (see above). This is illustrated by the observations that the accumulation of ammonium and glutamine during the day is accompanied by an accumulation of glycine and serine (Scheible et al., 2000
; Matt et al., 2001a), that when ammonium and glutamine accumulation are delayed in genotypes with decreased NIA activity there is a corresponding decrease of glycine and serine (Scheible et al., 1997b), and that when glycine and serine accumulation are decreased by growing plants in elevated [CO2] to decrease photorespiration the accumulation of ammonium and glutamine is also decreased (Geiger et al., 1998; Matt et al., 2001b). It was investigated whether supplying glycine or serine to leaves led to a decrease of NIA activity. Despite a several-fold increase in the internal pools, NIA activity was unaffected by glycine, but did decrease in response to serine (C Müller, R Morcuende, M Stitt, unpublished data).
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Malate leads to a marked decrease of the NIA transcript level and NIA activity |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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Assimilation of nitrate leads to alkalinization, which is counteracted by the synthesis of the counter-anion malate for export to the roots (see Introduction). Considerable amounts of malate nevertheless accumulate in leaves during the day and are remobilized during the night, revealing that malate export does not keep pace with the rate of nitrate assimilation (see above).
Malate was supplied to detached leaves in a range of conditions including the presence and absence of nitrate, in the light, after reilluminating predarkened leaves, and after supplying sucrose to dark-adapted leaves in the dark (Müller et al., 2001). In all cases, malate led to a marked decrease of NIA activity, which was associated with a strong decrease of the NIA transcript level. The decrease was found in response to changes of leaf malate levels within the range found during a diurnal cycle, indicating that it is physiologically relevant and represents a mechanism to decrease NIA activity when the rate of nitrate reduction exceeds the rate of malate export from the leaf.
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Low sugars lead to a strong and highly selective inhibition of NIA activity, and also inhibit amino acid biosynthesis |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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The experiments discussed in the previous sections were carried out with tobacco plants growing in high light and a 12 h or 14 h light period. As indicated above, no evidence was found for a major influence of sugar levels on NIA expression in any of these experiments. In the pioneering studies that established a role for sugars in the regulation of NIA expression (Cheng et al., 1992
; Vincentz et al., 1993), sucrose was added to plants after an extended dark pretreatment, which would have led to a severe depletion of sugars. It was decided to carry out a series of experiments to define the physiological range in which changes of sugars affect NIA expression and activity. These experiments would also show whether low sugars affect other processes in nitrogen metabolism or related aspects of carbon metabolism.
The first experiment was carried out in a model system in which the effects of sugars on NIA expression could be displayed, without interference due to the resulting changes in the rate of nitrate assimilation. To do this, the low-NIA activity tobacco line Nia30(145) was used. Nia30(145) carries a genomic NIA2 sequence which is expressed at low efficiency and contains four non-functional nia gene copies, which contain point mutations and are transcribed and translated to produce non-functional protein (Scheible et al., 1997a, b), providing a perfect reporter for NIA expression. As a consequence of the low NIA activity, the plants contain high nitrate, low glutamine and other amino acids, resulting in constitutive high expression of the defective nia genes and non-functional nia protein in a 14/10 h light/dark regime (Scheible et al., 1997a, b). The plants were grown on high nitrate in a 14/10 h light/dark regime, transferred to continuous darkness for several days and leaves then detached and supplied with a range of sucrose concentrations via the petiole in the dark (Klein et al., 2000). After transfer to darkness, there was a gradual decrease of NIA transcript, which was completely reversed by feeding sucrose in the dark. The decrease occurred even though nitrate remained high and glutamine and other amino acids very low. Interestingly, NIA protein declined more rapidly than NIA transcript, and this decrease was only partially restored by feeding sucrose indicating that protein synthesis or turnover is either more sensitive to falling sugar than translation, or that it is also affected by an additional signal related to light that is not required to restore transcript levels.
The main conclusions from these experiments were, therefore, that low sugars act (i) post-transcritionally and, when they are even lower, transcriptionally, to repress NIA and (ii) that this inhibition completely overrides signals from nitrogen metabolism, which would otherwise drive constitutive overexpression of NIA. A further important finding was that internal levels of 10–15 mM sugars suffice to fully restore the NIA transcript and partially restore NIA protein levels. This is lower than the levels found at any time during the diurnal cycle in plants growing in a ‘normal’ light/dark cycle at high or intermediate light intensities. Finally, no significant changes were seen for the levels of transcripts for PPC, PKC, ICDH-1 or GLN2, or the activities of the encoded enzymes. This reveals that NIA expression responds with special sensitivity to a decrease in the sugar supply.
Two further sets of experiments exploited changes in the light regime to investigate the regulatory interactions between nitrogen and carbon metabolism in wild-type tobacco. In one approach, wild-type tobacco was grown in a short day regime (6/18 h light/dark) (Matt et al., 1998). Normally, starch remobilization allows relatively high leaf sugar levels to be maintained throughout the night. When tobacco is grown in short-day conditions, however, leaf starch is depleted by the middle of the night and this is followed by a sharp decrease of the leaf sugar content. The NIA transcript level normally rises gradually during the night, following the recovery of leaf nitrate (see above). In short-day conditions, the NIA transcript recovers to high levels in the middle of the night and then, following the collapse of leaf sugar levels, falls to very low levels. Whereas NIA protein usually increases 2–3-fold after illumination, in short-day conditions this increase is strongly attenuated, presumably because the NIA transcript concentration is so low that it is limiting for the de novo synthesis of NIA protein. Indirect evidence for this interpretation is supplied by experiments mentioned above, where it was shown, using mutants, that the rate of increase of NIA activity after sudden illumination was proportional to the number of functional gene copies and, by implication, the concentration of functional NIA transcript. The low NIA activity in short-day conditions led to an accumulation of nitrate, low levels of amino acids, and a decreased protein and chlorophyll content. In a second approach, tobacco was grown in high (500 µmol m-2 s-1) and low (160 and 80 µmol m-2 s-1) irradiance in a 12/12 h light/dark regime (P Matt, M Stitt, unpublished data). Low light led to a small decrease of sugar levels, decreased Vmax NIA activity, accumulation of nitrate, low levels of amino acids, and a low protein and chlorophyll content.
These experiments revealed that carbohydrate status also affects nitrogen metabolism at sites downstream of nitrate assimilation. At the end of a long night, glutamate, aspartate and alanine were high, but the levels of all the minor amino acids except for asparagine were extremely low. Illumination reversed this, leading within 1 h to a decrease of glutamate whereas all the minor amino acids rose several fold (Matt et al., 1998). A similar reciprocal decrease of glutamate and increase of the minor amino acids was also seen after supplying sucrose to detached leaves in low light (Morcuende et al., 1998). These results indicate that there is a general inhibition of amino acid biosynthesis when sugars are low. This inhibition can develop and be reversed within 1–2 h.
To explore the interaction between sugar levels and nitrogen metabolism more systematically, an investigation into nitrogen metabolism in antisense RBCS tobacco ant3 and ant5 transformants was recently begun. These transformants have a 50–60% and a 95% reduction of Rubisco activity, respectively, compared to wild-type tobacco (P Matt, M. Stitt, unpublished results; see Stitt and Schulze, 1994, for background information). Decreased Rubisco activity led to a relatively small decrease of sugar levels, a large decrease of NIA activity, a very large decrease of amino acid levels, and a decrease of leaf protein that was larger than the decrease expected solely as a result of the decrease in the Rubisco content. These changes were marked in ant3, and larger in ant5. The decrease of amino acids was larger than the decrease of sugars, underlining the tight coupling between carbon and nitrogen metabolism.
The decrease of NIA activity was accompanied by a decrease of the NIA transcript in ant5 but not in ant3, confirming (see above for the presentation of the results in Klein et al., 2000) that small changes of sugars inhibit NIA expression via post-transcriptional mechanisms and larger changes lead to an inhibition of transcription. The activities of glutamine synthetase, aminotransferases and PPC were unaltered in both ant3 and ant5, confirming the special sensitivity of NIA to low sugar. GOGAT activity was only slightly decreased in ant3 but decreased by 50% in ant5 (P Matt, P Carillo, Y Gibon, unpublished data), which again highlights this enzyme as a second potential site for the regulation of central nitrogen metabolism. The levels of all the minor amino acid fell to very low levels, except asparagine. Analyses of the transcript levels for four enzymes in the shikimic acid pathway revealed that they did not decrease, indicating that amino acid biosynthesis is inhibited via post-transcriptional mechanisms. This is in agreement with the rapidity of the changes after switching light regimes or feeding sugars to detached leaves (see above).
Intriguingly, decreased expression of Rubisco led to a progressive increase of phosphoenolpyruvate and pyruvate, a decrease of acetyl coenzyme-A, citrate and, especially, 2-oxoglutarate (P Matt, M Stitt, unpublished data). The decrease of 2-oxoglutarate was accompainied by a decrease of glutamate, possibly because these metabolites are equilibrated or linked via aminotransferase reactions or glutamate dehydrogenase. The low glutamate may, in turn, act to restrict nitrogen flow into the amino acid biosynthesis pathways.
The increase of phosphoenolpyruvate and pyruvate shows that the decrease of 2-oxoglutarate is not simply due to a lower rate of photosynthesis and decreased levels of the phosphorylated intermediates, but involves an inhibition of pyruvate dehydrogenase, which converts pyruvate to AcCoA. One possible explanation would be that low Rubisco activity leads to an increase of ATP (Stitt and Schulze, 1994). If this includes an increase of ATP in the mitochondria, it would lead to phosphorylation and inactivation of pyruvate dehydrogenase (Randall et al., 1996). This proposal is supported by the observation that exposure to low light for 2 h (to decrease the rate of electron transport and decrease ATP levels) leads to an increase of 2-oxoglutarate in the ant3 transformants, and that exposure of wild-type plants to low [CO2] (which will lead to high ATP) for 2 h leads to a decrease of 2-oxoglutarate. This illustrates a further regulatory loop between photosynthesis, carbon metabolism and nitrogen metabolism, and emphasizes the importance of an appropriate balance between light absorbtion, electron transport and the use of ATP and NADP(H) in the dark reactions for the correct operation of ammonium assimilation and amino acid biosynthesis.
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Tight regulatory links between carbon and nitrogen metabolism lead to an unexpected scenario in which plants become carbon- and nitrogen-limited, with important consequences for secondary metabolism and fitness |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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The experiments described in the previous section show that nitrogen metabolism is very sensitive to an inhibition of photosynthesis. In plants growing in unfavourable light regimes or in transformants with low Rubisco activity, falling levels of sugars lead to decreased NIA activity, possibly to an inhibition of GOGAT activity, and result in a general inhibition of amino acid metabolism. As a result, the plants become carbon and nitrogen limited.
This close link between carbon and nitrogen metabolism has interesting consequences for plants that are adapting to unfavourable light regimes. On the one hand, nitrogen assimilation consumes carbon skeletons and energy, providing a rationale why nitrate reduction is subject to such strong regulation when sugar levels fall. Further, as starvation leads to protein and amino catabolism, a co-ordinate inhibition of amino acid biosynthesis will be essential to prevent a series of wasteful futile cycles. On the other hand, the finding that carbon depletion leads to a simultaneous nitrogen deficiency requires a re-evaluation of ideas about how plants adapt to shade, and what factors may determine fitness in unfavourable light regimes. First, some aspects of ‘shade adaptation’, including the decrease of leaf protein and chlorophyll, might not be just an adaptation to low light, but also, in part, a stress response, in the sense that the decrease which is greater than would occur if nitrogen assimilation were not inhibited is a side-effect of the lower rate of photosynthesis. Second, the decrease of amino acid levels is accompanied by a collapse of the levels of phenylpropanoids and a marked decrease of nicotine, both in low light and in the antisense RBCS transformants (P Matt, H-P Mock, M Stitt, unpublished results). A similar decrease of aromatic acids and phenylpropanoids is found in transformants where the levels of aromatic amino acids are lowered by overexpression of tryptophan carboxylase (Yao et al., 1995) or a small inhibition of plastid transketolase expression which has no effect on photosynthesis (Henkes et al., 2001). This indicates that the decline of phenylpropanoids in low light or the antisense RBCS transformants is a direct consequence of the inhibition of nitrogen metabolism and the resulting decrease of aromatic amino acids. Decreased levels of secondary metabolites will presumably impair the resistance to pathogens. Third, the finding that sugars and amino acids decrease when photosynthesis is inhibited means that the experimental basis of hypotheses about the relationship between nutrient status and the spectrum of secondary metabolites will need to be re-evaluated.
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Conclusion |
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TopAbstractIntroductionNitrogen metabolism undergoes...Excess NIA capacity allows...Nitrate leads to an...The expression and activity...Nitrate and ammonium...Glutamate exerts feedback...GOGAT and/or glutamate...A systematic search for...There is no evidence...Malate leads to a...Low sugars lead to...Tight regulatory links between...ConclusionReferences |
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As outlined in the Introduction, the assimilation of nitrate involves complex and ramifying interactions with many other aspects of the metabolic and cellular activities in a leaf cell. Correspondingly, nitrate assimilation is subject to multilayered regulation to co-ordinate it with other processes in the cell. The present studies throw light on several aspects of this process in tobacco, a C3 plant that synthesizes starch as well as sucrose in leaves. First, they reveal that tobacco contains excess capacity for nitrate reduction. This leads to a transient imbalance between the rate of nitrate reduction and upstream and downstream events in the first part of the light period, which triggers the complex diurnal changes seen in leaves of plants growing in high nitrate and high light. Second, the fluxes in nitrate and ammonium metabolism are closely integrated with organic acid metabolism, with groups of genes and enzyme activities being co-ordinately and reciprocally regulated to adjust fluxes as the physiological status changes during the diurnal cycle or in different growth conditions. Third, these results reveal that, whereas there are large transient changes in ammonium and glutamine, glutamate remains relative constant, indicating that GOGAT, possibly in conjunction with glutamate dehydrogenase, represents an important site for regulation. Fourth, a wide range of metabolites act as signals and triggers in this network. On the one hand, nitrate regulates the expression of a wide range of genes. This co-ordinates nitrate assimilation with the influx of nitrate and avoids deleterious side reactions that might occur if active NIA protein were to be present in the absence of the correct electron acceptor. It simultaneously activates ammonium metabolism and reprogrammes carbon metabolism to provide an appropriate spectrum of organic acids. Feedback regulation by a range of compounds including glutamate, cysteine, asparagine, serine, and malate co-ordinates the assimilation of nitrate with the use of reduced nitrogen for amino acid biosynthesis, with sulphur and carbon metabolism, with the cycling of ammonium in photorespiration, and with the regulation of cellular pH. The formation of 2-oxoglutarate, which is probably limiting for GOGAT activity in vivo, is regulated by a range of factors including nitrate, factors related to the accumulation of ammonium or nitrate and the sugar supply, and may be restricted in stress situations where low rates of Calvin cycle turnover may lead to overenergization and inhibit the conversion of PEP through to 2-oxoglutarate. Finally, low sugar leads to an overriding inhibition of NIA expression and nitrate assimilation, an inhibition of ammonium assimilation at GOGAT, and a general inhibition of amino acid biosynthesis, and a large decrease in the levels of the secondary metabolites that are synthesized from amino acids.
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Acknowledgments |
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This research was supported by the Deutsche Forschungsgemeinschaft.
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Notes |
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4 To whom correspondence should be addressed. Fax: +493315678101. E-mail: mstitt{at}mpimp\|[hyphen]\|golm.mpg.de
5 Present address: Unité de Nutrition Azotée des Plantes, INRA de Versailles, route de St Cyr, F-78026 Versailles cedex, France.
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 A. Neelam, T. Cassol, R. A. Mehta, A. A. Abdul-Baki, A. P. Sobolev, R. K. Goyal, J. Abbott, A. L. Segre, A. K. Handa, and A. K. Mattoo A field-grown transgenic tomato line expressing higher levels of polyamines reveals legume cover crop mulch-specific perturbations in fruit phenotype at the levels of metabolite profiles, gene expression, and agronomic characteristics J. Exp. Bot., June 1, 2008; 59(9): 2337 - 2346. [Abstract] [Full Text] [PDF]
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G. Tcherkez and M. Hodges How stable isotopes may help to elucidate primary nitrogen metabolism and its interaction with (photo)respiration in C3 leaves J. Exp. Bot., May 1, 2008; 59(7): 1685 - 1693. [Abstract] [Full Text] [PDF]
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 U. Lee, C. Wie, B. O. Fernandez, M. Feelisch, and E. Vierling Modulation of Nitrosative Stress by S-Nitrosoglutathione Reductase Is Critical for Thermotolerance and Plant Growth in Arabidopsis PLANT CELL, March 1, 2008; 20(3): 786 - 802. [Abstract] [Full Text] [PDF]
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S. Rasmussen, A. J. Parsons, K. Fraser, H. Xue, and J. A. Newman Metabolic Profiles of Lolium perenne Are Differentially Affected by Nitrogen Supply, Carbohydrate Content, and Fungal Endophyte Infection Plant Physiology, March 1, 2008; 146(3): 1440 - 1453. [Abstract] [Full Text] [PDF]
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 K. Okazaki, N. Oka, T. Shinano, M. Osaki, and M. Takebe Differences in the Metabolite Profiles of Spinach (Spinacia oleracea L.) Leaf in Different Concentrations of Nitrate in the Culture Solution Plant Cell Physiol., February 1, 2008; 49(2): 170 - 177. [Abstract] [Full Text] [PDF]
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D. S. Skopelitis, N. V. Paranychianakis, A. Kouvarakis, A. Spyros, E. G. Stephanou, and K. A. Roubelakis-Angelakis The Isoenzyme 7 of Tobacco NAD(H)-Dependent Glutamate Dehydrogenase Exhibits High Deaminating and Low Aminating Activities in Vivo Plant Physiology, December 1, 2007; 145(4): 1726 - 1734. [Abstract] [Full Text] [PDF]
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A. Whittaker, T. Martinelli, J. M. Farrant, A. Bochicchio, and C. Vazzana Sucrose phosphate synthase activity and the co-ordination of carbon partitioning during sucrose and amino acid accumulation in desiccation-tolerant leaf material of the C4 resurrection plant Sporobolus stapfianus during dehydration J. Exp. Bot., October 1, 2007; 58(13): 3775 - 3787. [Abstract] [Full Text] [PDF]
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B. G. Forde and P. J. Lea Glutamate in plants: metabolism, regulation, and signalling J. Exp. Bot., July 1, 2007; 58(9): 2339 - 2358. [Abstract] [Full Text] [PDF]
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 C. Tian, E. Chikayama, Y. Tsuboi, T. Kuromori, K. Shinozaki, J. Kikuchi, and T. Hirayama Top-down Phenomics of Arabidopsis thaliana: METABOLIC PROFILING BY ONE- AND TWO-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY AND TRANSCRIPTOME ANALYSIS OF ALBINO MUTANTS J. Biol. Chem., June 22, 2007; 282(25): 18532 - 18541. [Abstract] [Full Text] [PDF]
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A. K. Mattoo, A. P. Sobolev, A. Neelam, R. K. Goyal, A. K. Handa, and A. L. Segre Nuclear Magnetic Resonance Spectroscopy-Based Metabolite Profiling of Transgenic Tomato Fruit Engineered to Accumulate Spermidine and Spermine Reveals Enhanced Anabolic and Nitrogen-Carbon Interactions Plant Physiology, December 1, 2006; 142(4): 1759 - 1770. [Abstract] [Full Text] [PDF]
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D. S. Skopelitis, N. V. Paranychianakis, K. A. Paschalidis, E. D. Pliakonis, I. D. Delis, D. I. Yakoumakis, A. Kouvarakis, A. K. Papadakis, E. G. Stephanou, and K. A. Roubelakis-Angelakis Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine PLANT CELL, October 1, 2006; 18(10): 2767 - 2781. [Abstract] [Full Text] [PDF]
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J. Persson, P. Gardestrom, and T. Nasholm Uptake, metabolism and distribution of organic and inorganic nitrogen sources by Pinus sylvestris J. Exp. Bot., August 1, 2006; 57(11): 2651 - 2659. [Abstract] [Full Text] [PDF]
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P. Walch-Liu, L.-H. Liu, T. Remans, M. Tester, and B. G. Forde Evidence that L-Glutamate Can Act as an Exogenous Signal to Modulate Root Growth and Branching in Arabidopsis thaliana Plant Cell Physiol., August 1, 2006; 47(8): 1045 - 1057. [Abstract] [Full Text] [PDF]
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 H. F. Causin, T. W. Rufty, and J. F. Reynolds Gas exchange and carbon metabolism in two Prosopis species (Fabaceae) from semiarid habitats: effects of elevated CO2, N supply, and N source Am. J. Botany, May 1, 2006; 93(5): 716 - 723. [Abstract] [Full Text] [PDF]
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U. S. Lea, M.-T. Leydecker, I. Quillere, C. Meyer, and C. Lillo Posttranslational Regulation of Nitrate Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas Transcriptional Regulation Has Only Minor Influence Plant Physiology, March 1, 2006; 140(3): 1085 - 1094. [Abstract] [Full Text] [PDF]
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Z. YANG and D. J. MIDMORE A Model for the Circadian Oscillations in Expression and Activity of Nitrate Reductase in Higher Plants Ann. Bot., November 1, 2005; 96(6): 1019 - 1026. [Abstract] [Full Text] [PDF]
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C. Dutilleul, C. Lelarge, J.-L. Prioul, R. De Paepe, C. H. Foyer, and G. Noctor Mitochondria-Driven Changes in Leaf NAD Status Exert a Crucial Influence on the Control of Nitrate Assimilation and the Integration of Carbon and Nitrogen Metabolism Plant Physiology, September 1, 2005; 139(1): 64 - 78. [Abstract] [Full Text] [PDF]
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S. J. Cookson, L. E. Williams, and A. J. Miller Light-Dark Changes in Cytosolic Nitrate Pools Depend on Nitrate Reductase Activity in Arabidopsis Leaf Cells Plant Physiology, June 1, 2005; 138(2): 1097 - 1105. [Abstract] [Full Text] [PDF]
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D. Gonzalez-Ballester, A. de Montaigu, J. J. Higuera, A. Galvan, and E. Fernandez Functional Genomics of the Regulation of the Nitrate Assimilation Pathway in Chlamydomonas Plant Physiology, February 1, 2005; 137(2): 522 - 533. [Abstract] [Full Text] [PDF]
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E. Urbanczyk-Wochniak and A. R. Fernie Metabolic profiling reveals altered nitrogen nutrient regimes have diverse effects on the metabolism of hydroponically-grown tomato (Solanum lycopersicum) plants J. Exp. Bot., January 1, 2005; 56(410): 309 - 321. [Abstract] [Full Text] [PDF]
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U. DRUEGE, S. ZERCHE, and R. KADNER Nitrogen- and Storage-affected Carbohydrate Partitioning in High-light-adapted Pelargonium Cuttings in Relation to Survival and Adventitious Root Formation under Low Light Ann. Bot., December 1, 2004; 94(6): 831 - 842. [Abstract] [Full Text] [PDF]
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W.-R. Scheible, R. Morcuende, T. Czechowski, C. Fritz, D. Osuna, N. Palacios-Rojas, D. Schindelasch, O. Thimm, M. K. Udvardi, and M. Stitt Genome-Wide Reprogramming of Primary and Secondary Metabolism, Protein Synthesis, Cellular Growth Processes, and the Regulatory Infrastructure of Arabidopsis in Response to Nitrogen Plant Physiology, September 1, 2004; 136(1): 2483 - 2499. [Abstract] [Full Text] [PDF]
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R. Wang, R. Tischner, R. A. Gutierrez, M. Hoffman, X. Xing, M. Chen, G. Coruzzi, and N. M. Crawford Genomic Analysis of the Nitrate Response Using a Nitrate Reductase-Null Mutant of Arabidopsis Plant Physiology, September 1, 2004; 136(1): 2512 - 2522. [Abstract] [Full Text] [PDF]
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K. Takei, N. Ueda, K. Aoki, T. Kuromori, T. Hirayama, K. Shinozaki, T. Yamaya, and H. Sakakibara AtIPT3 is a Key Determinant of Nitrate-Dependent Cytokinin Biosynthesis in Arabidopsis Plant Cell Physiol., August 15, 2004; 45(8): 1053 - 1062. [Abstract] [Full Text] [PDF]
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 S. Rachmilevitch, A. B. Cousins, and A. J. Bloom Nitrate assimilation in plant shoots depends on photorespiration PNAS, August 3, 2004; 101(31): 11506 - 11510. [Abstract] [Full Text] [PDF]
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J. Price, A. Laxmi, S. K. St. Martin, and J.-C. Jang Global Transcription Profiling Reveals Multiple Sugar Signal Transduction Mechanisms in Arabidopsis PLANT CELL, August 1, 2004; 16(8): 2128 - 2150. [Abstract] [Full Text] [PDF]
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V. Kumar, D. J. Mills, J. D. Anderson, and A. K. Mattoo An alternative agriculture system is defined by a distinct expression profile of select gene transcripts and proteins PNAS, July 20, 2004; 101(29): 10535 - 10540. [Abstract] [Full Text] [PDF]
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A. M. Borland and T. Taybi Synchronization of metabolic processes in plants with Crassulacean acid metabolism J. Exp. Bot., June 1, 2004; 55(400): 1255 - 1265. [Abstract] [Full Text] [PDF]
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G. Noctor, C. Dutilleul, R. De Paepe, and C. H. Foyer Use of mitochondrial electron transport mutants to evaluate the effects of redox state on photosynthesis, stress tolerance and the integration of carbon/nitrogen metabolism J. Exp. Bot., January 1, 2004; 55(394): 49 - 57. [Abstract] [Full Text] [PDF]
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Y. Sonoda, A. Ikeda, S. Saiki, T. Yamaya, and J. Yamaguchi Feedback Regulation of the Ammonium Transporter Gene Family AMT1 by Glutamine in Rice Plant Cell Physiol., December 15, 2003; 44(12): 1396 - 1402. [Abstract] [Full Text] [PDF]
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F. Carrari, A. Nunes-Nesi, Y. Gibon, A. Lytovchenko, M. E. Loureiro, and A. R. Fernie Reduced Expression of Aconitase Results in an Enhanced Rate of Photosynthesis and Marked Shifts in Carbon Partitioning in Illuminated Leaves of Wild Species Tomato Plant Physiology, November 1, 2003; 133(3): 1322 - 1335. [Abstract] [Full Text] [PDF]
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A. Stepansky and G. Galili Synthesis of the Arabidopsis Bifunctional Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase Enzyme of Lysine Catabolism Is Concertedly Regulated by Metabolic and Stress-Associated Signals Plant Physiology, November 1, 2003; 133(3): 1407 - 1415. [Abstract] [Full Text] [PDF]
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X. Zhu and G. Galili Increased Lysine Synthesis Coupled with a Knockout of Its Catabolism Synergistically Boosts Lysine Content and Also Transregulates the Metabolism of Other Amino Acids in Arabidopsis Seeds PLANT CELL, April 1, 2003; 15(4): 845 - 853. [Abstract] [Full Text] [PDF]
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C. H. Foyer, M. Parry, and G. Noctor Markers and signals associated with nitrogen assimilation in higher plants J. Exp. Bot., January 3, 2003; 54(382): 585 - 593. [Abstract] [Full Text] [PDF]
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B. J. Miflin and D. Z. Habash The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops J. Exp. Bot., April 15, 2002; 53(370): 979 - 987. [Abstract] [Full Text] [PDF]
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