Recent research shows that signals derived from nitrate are involved in triggering widespread changes in gene
expression, resulting in a reprogramming of nitrogen and carbon metabolism to facilitate the uptake and assimilation of nitrate, and to initiate accompanying changes in carbon metabolism. These nitrate-derived signals interact with signals generated further downstream in nitrogen
metabolism, and in carbon metabolism. Signals derived from internal and external nitrate also adjust root growth and architecture to the physiological state of the plant, and the distribution of nitrate in the environment.
Addresses
Botanisches Institut, In Neuenheimer Feld 360, 69120 Heidelberg, Germany; e-mail: mstitt@botanik1.bot.uni-heidelberg.de
Current Opinion in Plant Biology1999, 2:178–186 http://biomednet.com/elecref/1369526600200178 © Elsevier Science Ltd ISSN 1369-5266
Abbreviation
NIA nitrate reductase
Introduction
Nitrate fertilisation leads to higher levels of amino acids and protein and increased growth [1,2,3•,4•,5], and also to changes in carbon metabolism including increased levels of organic acids and decreased levels of starch [6••,7••], to changes in phytohormone levels [1,2], and to changes in allocation and phenology including a decreased root:shoot ratio [6••], altered root architecture [1,6••,8•], and delayed flowering [5], tuber initiation and senescence [1,2]. These far-reaching changes underline the important role played by the signalling mechanisms that regulate metabolism and development in response to the availability of nitrogen.
Signals might be derived from nitrate itself, from metabo-lites formed during nitrate assimilation, or, more indirectly, as a result of changes in other cell constituents or the over-all rate of growth. In addition, signals originating from downstream events may exert negative feed-back regula-tion on earlier steps in nitrate acquisiregula-tion. In a given situation, it is likely that several signals interact to condi-tion the observed response. In microbes, nutrients typically induce genes required for their uptake and metabolism. It has been known since the early 1970’s that nitrate uptake [1,3•,4•,9] and nitrate reductase (NIA) activ-ity [2,10] increase after adding nitrate. The following review asks whether signals derived from nitrate regulate metabolic and physiological processes in higher plants, and then considers how nitrate-signalling interacts with signals generated further downstream in metabolism.
Nitrate as a regulator of genes for
primary metabolism
Using mutants with low expression of NIA, the external and internal nitrate concentration can be varied indepen-dently of the rate of nitrate assimilation and the resulting changes in the levels of downstream metabolites and growth rate [6••,7••,11,12,13•,14,15••]. Complementary approaches to identify nitrate-regulated processes include investigating the effect of nitrate on transcript levels in presence of inhibitors of nitrate or ammonium metabolism [12,16••], and investigation of transcripts that increase rapidly after adding nitrate [17–19,20••]. Table 1 sum-marises genes shown to be induced by nitrate and Figure 1 summarises the relevant metabolic pathways.
Nitrate induces genes encoding the high (NRT2) [13•,14,15••,21] and low (NRT1) [14,15••,22,23] affinity nitrate uptake systems, for nitrate reductase (NIA) [7••,11,12,16••,18], nitrite reductase (NII) [7••,12,16••], and the enzymes required for ammonium assimilation via the GOGAT pathway (GLN2, GLN1, GLU) [7••,17,24•]. The increase in transcript is accompanied by increased rates of nitrate uptake [13•,15••,21,25•], increased NIA protein and activity [7••,11,12], and increased activity of NII and glutamine synthetase [7••].
Nitrate assimilation requires synthesis of organic acids, especially α-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 alkalisation (Figure 1). Nitrate leads to a marked increase of transcripts encoding proteins involved in the synthesis of these organic acids (PKc, PPC, CS, ICDH-1; see Table 1) [7••], an increase in the activity of the corresponding enzymes [7••], and an accumulation of malate and α-oxog-lutarate [7••]. Fumarase, which catalyses a reaction in a section of the tricarboxylic acid cycle that is not required during nitrate assimilation, is not induced by nitrate (A Krapp, W-R Scheible, M Stitt, unpublished data).
Increased synthesis of organic acids requires diversion of carbon from carbohydrate synthesis. Accordingly, nitrate represses AGPS, encoding the regulatory subunit of ADP-glucose pyrophosphorylase, a key enzyme in the starch synthesis pathway [7••], leading to decreased activity of ADP-glucose pyrophosphorylase and a marked depletion of starch ([7••]; W-R Scheible, A Krapp, M Stitt, unpublished data). Interestingly, sucrose phosphate synthase (SPS) is not repressed by nitrate [7••], indicating that sucrose production continues. This may be important, because amino acid export and utili-sation requires continued synthesis of sucrose. These results open up new strategies to engineer carbon parti-tioning to starch accumulation.
Nitrate and nitrite reduction consume NADH in the cyto-plasm and reduced ferredoxin in the plastid, respectively. Reducing equivalents can be provided directly or indirect-ly by photosynthetic electron transport in leaves in the light, but in the dark and in non-photosynthetic organs they are supplied by respiratory metabolism. Addition of nitrate leads to rapid induction of relevant genes in roots,
including the oxidative pentose phosphate pathway enzyme 6-phosphogluconate dehydrogenase [20••], ferre-doxin [26••] and ferredoxin:NADP oxidoreductase [19].
Thus, nitrate leads to rapid changes in the levels of a wide range of transcripts encoding enzymes in nitrogen and car-bon metabolism. This allows a reprogramming of nitrogen
Table 1
Genes known to be regulated by nitrate-mediated signalling.
Physiological function Gene Protein Regulated in leaf Regulated in root References
Nitrate uptake NRT1 high affinity NO3transporter + [13•,14,15••,21]
NRT2 low affinity NO3transporter + [14,15••,22,23]
Nitrate assimilation NIA nitrate reductase + + [7••,11,12,16••,18]
NII nitrite reductase + + [7••,11,16••]
Ammonium assimilation GLN1 plastid glutamine synthetase + + [7••,17]
GLN2 cytosolic glutamine synthetase + + [7••,17,24•]
GLU GOGAT + + [7••]
Organic acid metabolism PPC phosphoenolpyruvate carboxylase + + [7••]
PKc cytosolic pyruvate kinase + + [7••]
CS citrate synthase + + [7••]
ICDH1 NADP-isocitrate dehydrogenase + + [7••]
Redox metabolism PGD 6-phosphogluconate dehydrogenase ? + [20••]
FNR ferredoxin:NADP oxidoreductase ? + [19]
FD ferredoxin ? + [26••]
Starch synthesis AGPS ADP-glucose pyrophosphorylase
(regulatory subunit) – ? [7••]
Figure 1
Nitrate-regulated genes in primary metabolism. Nitrate induces genes required for nitrate accumulation, ammonium accumulation, the synthesis of carbon acceptors like α-oxoglutarate, the synthesis of organic acids like malate to maintain pH balance, and, in nonphotosynthetic tissues, also induces genes required to generate redox equivalents during respiratory metabolism. In parallel, nitrate represses genes required for starch synthesis. For abbreviations, see Table 1.
NO3
CO2
NO2 NH3 Gln
Redox equivalents
Malate
OA
Pyr
PEP AcCoA Citrate
Starch
αOG
Sucrose Glu Amino acids
PGD FD FNR
CS ICDH
NIA NII GLU
GLN1 GLN2
PK
SPS PPC
AGPS
and carbon metabolism, to facilitate the assimilation of nitrate and its incorporation into amino acids. It will also contribute to the regulation of metabolism in limiting nitro-gen [7••,27•] and in elevated CO
2, where faster growth often leads to a marked decrease of nitrate [27•,28], in par-ticular it may explain why starch accumulates even though sugars often remain low in these conditions. Further work is needed to investigate if nitrate-derived signals affect the post-translational regulation of pathway metabolism, and whether further cellular processes are also regulated by nitrate, including the poising of photosynthetic electron transport, starch re-mobilisation, storage protein expres-sion, and the synthesis of secondary metabolites.
Interaction with downstream events in
nitrogen metabolism
Nitrate-signalling interacts with signals generated further downstream in nitrogen metabolism and carbon metabo-lism. This is illustrated by the three general observations. First, whereas nitrate addition leads to a transient induc-tion of genes involved in nitrate uptake and assimilainduc-tion and organic acid synthesis in wild-type plants, it results in a sustained overexpression of these genes in low NIA mutants [7••,12,13•]. Secondly, whereas wild-type plants show pronounced diurnal changes of transcripts levels encoding enzymes involved in nitrate, ammonium and organic acid biosynthesis and diurnal changes of the corre-sponding enzyme activities that correlate with diurnal fluctuations of the sugar, amino acid and organic acid pools ([29•]; A Krapp, W-R Scheible, M Stitt, unpublished data), NIA-deficient mutants have constitutive and high levels of these transcripts and enzyme activities throughout the day and night [11,29•]. Third, addition of glutamine, asparagine or other amino acids inhibits nitrate uptake [3•,4•] and nitrate assimilation [12].
In prokaryotes and fungi, glutamine and α-oxoglutarate (see below) play a dominating role in regulating metabolism. The metabolites involved in downstream nitrogen-sig-nalling have not been characterised as clearly in plants. In leaves, the endogenous level of ammonium or glutamine often correlates negatively with the NIA transcript level [7••,12,16••,29•,30•], and ammonium or glutamine addition typically lead to a decrease of the transcripts for NRT2 [13•,14,21], NRT1 [15••,23], NIA [12,16••,29•,31] and NII [31]. Inhibitor experiments indicate that glutamine rather than ammonium is the metabolite responsible for the repression of NIA [12]. Glutamine and asparagine also repressNIAin maize roots [32•].
Two recent studies, however, point towards more a com-plicated situation. Split-root experiments with castor bean plants indicate that feed-back inhibition of nitrate uptake involves a shoot-derived signal that does not require increased levels of amino acids in the phloem [33•]. In a new approach to manipulate glutamine levels in vivo, where glu-deficient Arabidopsis mutants were transferred from high to low CO2, the resulting
over-accumulation of glutamine was not accompanied by a decrease of NIA transcript [34••]. As there was a small decrease of leaf nitrate, it was proposed that the exoge-nous glutamine used in previous experiments acts indirectly, by inhibiting nitrate uptake [34••]. Alternative explanations would be that NIAis repressed by a specif-ic glutamine pool whspecif-ich is not affected in the glu mutants, or that the increase of glutamine is compensat-ed for by changes in other unidentificompensat-ed metabolite pools.
Nitrogen metabolism is complicated in leaves of C-3 plants by fluxes of ammonium around the photorespira-tory cycle, which can be 10-fold higher than the net rate of nitrate assimilation. It will be interesting to learn how changes of compounds related to ammonium assimila-tion can be used as a reliable indicators for nitrogen status, when their turnover is dominated by an unrelated process that reflects the rate of photosynthesis and the stomatal aperture.
Interaction with sugar signalling
In many cases, sugars exert complementary effects to nitrate on gene expression. Sugars induce NRT1 and NRT2 [15••], NIA, GLN1, pyruvate kinase, PPC, and ICDH1[35,36], and stimulate the post-translational acti-vation of NIA [2,32•,37]. The complementary effects of nitrate and sugars are illustrated by the demonstration that both have to be supplied to achieve high rates of nitrate assimilation and amino acid synthesis in detached leaves [38•]. High sucrose stimulates the conversion of nitrate to glutamine and, especially, the conversion of glycerate-3-phosphate (the immediate product of photo-synthesis) to α-oxoglutarate [38•]. When sugars are low they exert a strong negative regulation on NIA expres-sion, that over-rides the signals derived from nitrogen metabolism [39•]. It is not yet known whether these effects of sugars on nitrate metabolism are mediated via the currently discussed signalling mechanisms involving hexokinase, or by other means [35,36].
Interaction with pH
Nitrate and the regulation of shoot–root
allocation and root architecture
It has been known for a long time that root growth and architecture is modified by nitrogen fertilisation. High nitrate preferentially inhibits root growth, leading to a decrease of the root:shoot ratio [1,41] and decreasing the frequency of lateral roots [1,42]. Local application of nitrate to roots of nitrogen-limited plants, on the other hand, leads to localised proliferation of lateral roots [43] (Figure 2). As a consequence, nitrogen limited plants for-age a larger soil volume for nitrogen-containing nutrients, and direct their root growth in response to the spatial dis-tribution of nutrients in the soil. Recent research is revealing that signals derived from nitrate trigger these adaptive changes in root growth and architecture.
NIA-deficient tobacco transformants growing on high nitrate develop an abnormally high shoot:root ratio [6••], even though they are severely deficient for amino acids and protein. The inhibition of root growth involves a decrease in the number of growing lateral roots [8•], correlates with nitrate accumulation in the plant, and was shown, in split-root experiments, to be due to an unidentified signal generated in the shoot [6••]. High external nitrate also leads to a decrease in the number of growing lateral roots in Arabidopsis, especially in NIA-deficient mutants [44••,45••].
When NIA-deficientArabidopsisis grown on low nitrate, and nitrogen supplied to a small region of the root, nitrate leads to proliferation of lateral roots at this site, whereas other nitrogen sources were ineffective [44••]. Similar results were obtained in split-root experiments with NIA-deficient tobacco, where it was also shown that amino acids and proteins decreased in the root sector where growth had been stimulated [6••].
Thus, signals derived from internal and external nitrate interact to modulate root growth and architecture, and allow efficient exploitation of the nutrient supply in the atmosphere. The role of nitrate in regulating other impor-tant whole plant processes like flowering and senescence still has to be investigated. It is also intriguing that hyper-nodulating mutants have been identified where the increased nodule number is due to loss of a shoot-depen-dent nitrate-repression of module formation, and also show a pleiotropic increase of root lateral frequency when they are grown in the absence of Rhizobium[46–49].
Mechanisms for nitrogen signalling in
prokaryotes, fungi and plants
The molecular mechanism(s) of nitrate-sensing in higher plants are still unknown. It can be anticipated, however, that several mechanisms are involved. The cytosolic nitrate concentration is maintained constant [50,51•] whereas high concentrations of nitrate are accumulated in and re-mobilised from the vacuole. In leaves, the levels of nitrate-induced transcripts like NIAdecrease after illumi-nation, and re-fertilisation with high nitrate concentrations later in the light period leads to re-induction of NIA, even
though the overall pool of nitrate remains high throughout the day [30•]. It, therefore, appears necessary to postulate separate sensing systems to monitor incoming nitrate, to maintain a nitrate homeostasis in the cytosol and, possibly, to monitor vacuolar nitrate.
In bacteria, the best-characterised nitrate-sensing mecha-nism regulates an operon that contains genes for a set of electron transport components and an anaerobiosis-specif-ic NIA whanaerobiosis-specif-ich catalyses electron transfer to a nitrate terminal acceptor. Very low concentrations of extracellular nitrate are sensed via two functionally overlapping sensors NARX/NARQ and NARL/NARP [52,53]. Nitrate or nitrite lead to transfer of phosphate from the autophospho-rylating histidine kinase sensor components (NARX, NARQ) to the corresponding DNA-binding response ele-ment (NARL, NARP). Homologous systems occur in higher plants, but it is not known any if any of them rep-resent functional homologs for nitrate-sensing. Intriguingly (see below), several are induced by nitrate.
Carbon–nitrogen interactions are regulated via sensing of downstream metabolites in E. coli. Glutamine and α-oxog-lutarate are sensed in E. coli via a cascade involving the bifunctional uridyl transferase/uridyl removing enzyme (UT–UR), the regulatory PIIprotein that is modulated by uridenylation and deuridenylation, the bifunctional kinase/phosphatase NRII and the transcription factor NR1. This cascade exerts transcriptional and post-translational regulation on glutamine synthetase. UT–UR and PII prob-ably represent the sensor, as they are constitutively expressed [54], and their activity is regulated by metabo-lites [55,56]. There is recent evidence in E. coliand other species [57,58] including cyanobacteria [59], however, for a
Figure 2
Root architecture is regulated by an interaction between shoot nitrate, which acts via an uncharacterised long distance signal to inhibit lateral root growth, and local nitrate levels around the root, which stimulate lateral root growth.
NO3 NO3
Local NO3
PII homolog that is transcriptionally regulated by metabo-lites. The cyanobacterial PII homolog is post-translationally regulated by phosphorylation instead of uridenylation, and may be involved in the post-transcriptional regulation of nitrate uptake [60]. A nuclear-encoded PII homolog (GLB1) was recently identified in Arabidopsis[61••]. GLB1lacks the uridinylation site at Tyr-51 and, in analogy to other newly discovered PII homologs, is induced by sucrose and repressed by glutamine, asparagine and aspartate. GLB1 is located in the plastid, indicating a role in sensing or regu-lating ammonium assimilation in the plastid, and raising fascinating problems with respect to the communication between the plastid and the nucleus.
In filamentous fungi [62,63], nitrate reductase (here desig-nated NIT3) expression is regulated by two positive acting transcription factors (NIT4 and NIT2 in Neurospora crassa, NIRA and AREA in Aspergillus nidulans). NIT4/NIRA is a member of the GAIA family and binds to an unusual asym-metrical nucleotide sequence [64] to mediate a pathway-specific induction of nitrate uptake and NIT3. NIT2/AREA is member of the GATA family of transcrip-tion factors and acts positively in the absence of preferred nitrogen sources like ammonium and glutamine to induce genes required to utilise alternative nitrogen sources like nitrate, and to catabolise amino acids. When preferred nitrogen sources are present these pathways are repressed, because NIT2/AREA is inactivated. An NIT2 homolog positively regulates expression of nitrate transport and assimilation in the absence of ammonium in Chlamydomonas [65]. The function of a tobacco NIT2 homolog [66] is still unclear. Recently, NIT2 binding sequences have been reported in the spinach NII promotor [67••], and two fur-ther putative transcription factors designated RGA1 and RGA2 have been isolated via functional complementation of a GLN3-deficient yeast mutant (the yeast homolog of AREA/NIT2) [68••]. As discussed in [69], these two genes are identical with GAIand RGA, that negatively modulate gibberellin-induced changes during development.
The repertoire of sensing mechanisms will probably grow. In Chlamydomonas, the nitrate-induction of genes encoding nitrate and nitrite transporter components is modified by constitutive expression of NIA [70], probably due to regu-lation exerted by nitrite (E Frenandez, personal communication). Two novel regulatory genes RGA1 and RGA2 required for the ammonium modulation of NIA expression have also been identified in Chlamydomonas [71]. One fascinating recent development is the realisation that glutamine-tRNA isoform acts as a indicator for nitro-gen source quality in yeast, and regulates the uptake and use of non-preferred nitrogen sources and sporulation inde-pendently of changes in the rate of protein synthesis [72•]. Transplant proteins can also act in yeast [73–76].
Recent studies have elucidated novel downstream sig-nalling components in nitrate-sigsig-nalling pathways in higher plants. It has been demonstrated, using reverse genetics,
that a nitrate-induced transcription factor ANR1 plays an important role in the stimulation of lateral root growth by localised application of nitrate, and in the inhibition of lat-eral root growth by high nitrate [44••]. ANR1 encodes a MADS-box transcription factor, a family of proteins that act as molecular switches during development, and represents the first example where expression is environmentally reg-ulated. The stimulatory and inhibitory effects of nitrate on lateral root growth are exerted just after the lateral initials emerge from the primary root [45••], and are blocked in the axr4 auxin-resistant mutants [45••], indicating a fascinating cross-talk with hormonal regulation of root development. Lateral root growth clearly provides an exciting model sys-tem for molecular analysis of an interplay between an endogenous developmental programme and signals that modulate it in response to the physiological state of the plant and the environmental conditions.
Recent work on nitrate-induced gene expression in maize [24•,77••,78] is providing information about downstream components in two further nitrogen-signalling pathways. Nitrate addition to detached leaves rapidly induces NIA, NII, GS2, ferredoxin dependent-GOGAT and NADH-dependent-GOGAT [24•], whereas PPC (which in a C-4 plant is essential for photosynthesis as well as net organic acid synthesis) is not induced by nitrate addition to detached leaves, but is induced when nitrate or ammonium are added to the roots of whole plants [77••]. The nitrate-specific induction of the nitrate assimilation pathway in leaves may involve calcium and protein phosphorylation [24•] via protein kinase C [78]. Induction of PPCin whole plants, on the other hand, is associated with increased lev-els of cytokinins in the xylem sap and an increase of the transcript for a cytokinin induced protein (pZmCIP1) in leaves [77••]. The increase of the PPCand pZmCIP1 tran-scripts could be mimicked by feeding cytokinins to detached leaves [77••]. pZmCIP1 encodes a nitrogen- and cytokinin-induced homolog of the response element of bac-terial two component signalling systems. A small family of response regulator proteins (ARR3–ARR7) showing a simi-lar cytokinin- and nitrate-dependent expression has recently been identified in Arabidopsis[79••]. It will be fas-cinating to learn which endogenous ligands interact with these cytokinin and nitrogen-induced sensors, and what phenotypes result when their expression is disrupted.
organism. Although progress will certainly draw on analyses of nitrogen-signalling in microbes, appropriate and novel approaches will also be needed in higher plants. The recent advances in identifying a wider range of nitrate-regulated genes and nitrate-dependent changes in plant growth and development provide important tools to address these problems, as will the application of multi-parallel gene expression, and the exploitation of insertion mutants and other novel sources of genetic variability including activation-tagged lines and the natural biological diversity available in ecotypes and inbred recombinant lines.
Conclusions
Nitrate sensing provides one of the mechanisms whereby plants sense nitrogen availability in the environment and their internal nitrogen status. At a cellular level, it leads to reprogramming of metabolism to allow nitrate to be assimilated and incorporated into organic compounds, and at a whole plant level it modulates allocation and development to allow root growth and nutrient uptake to respond to spatial and temporal fluctuations in the avail-ability of nitrate. We can expect that molecular analysis of nitrate-signalling will provide further important insights into the networking of metabolism, and into the mecha-nisms whereby plant development is modulated by response to changes in the environment.
Acknowledgements
Support for research in the author’s laboratory is acknowledged from the Deutsche Forschungsgemeinschaft (Sti78 2/2; SFB 199; Schwerpunktprogramme ‘Metabolism and growth of plants in elevated carbon dioxide concentrations’, and the European Commission (BIO4 CT97 2231). I am grateful to Brian Forde, Werner Kaiser and Ton Bisseling for discussions, and to Brian Forde and Alain Gojon for making unpublished results available to me.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
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Increased expression of NIA leads to decreased nitrate uptake, whereas decreased expression leads to accumulation of nitrate in the plants. These results indicate that a downstream metabolite of nitrate metabolism repress-es nitrate uptake, and provide direct evidence that changrepress-es in the rate of nitrate assimilation in the leaves have consequences for the rate of nitrate uptake in the roots. Possible molecular mechanisms for this interaction are presented in [15••].
26 Matsumara T, Sakaibara H, Nakano R, Kimata Y, Sugiyama T, Hase T:
•• A nitrate-inducible ferredoxin in maize roots: genomic organisation and differential expression of two non-photosynthetic ferredoxin apoproteins.Plant Physiol1997, 114:653-660.
Reduction of nitrate to ammonium in the roots requires reduced ferredoxin for the plastid-localised NII step. This paper shows that nitrate induces a non-photosynthetic ferredoxin genes in maize roots. Together with [20••], it shows that nitrate re-programmes root cells to increase the delivery of redox equivalents in the appropriate form to support nitrate assimilation. 27 Geiger M, Haake V, Ludewig F, Sonnewald U, Stitt M: Influence of
• nitrate and ammonium nitrate supply on the response of photosynthesis, carbon and nitrogen metabolism, and growth to elevated carbon dioxide in tobacco.Plant Cell Environ 1999, in press.
This study of the effect of elevated CO2on transcripts and activities of
enzymes in nitrogen and carbon metabolism, on metabolite levels and growth of plants growing at several nitrogen supplies shows that acclimation of photosynthesis the increased accumulation of starch in elevated CO2are
triggered by changes in the nitrogen status, including nitrate. This challenges the wide-spread view that these changes in carbon metabolism are caused by sugar-regulation of gene expression.
28 Stitt M, Krapp A: The molecular physiological basis for the interaction between elevated carbon dioxide and nutrients.Plant Cell Environ1999, in press.
29 Scheible W-R, Gonzalez-Fontes A, Morcuende R, Lauerer M,
• Geiger M, Glaab J, Gojon A, Schulze E-D, Stitt M: Tobacco mutants with a decreased number of functional niagenes compensate by
modifying the diurnal regulation of transcription,
post-translational modification and turnover of nitrate reductase. Planta
1997, 203:304-319.
It is well established in several species that mutants with a 50–90% decrease in maximum NIA activity grow at similar rates to wild-type plants. This study of tobacco mutants shows that they compensate by altering the diurnal regulation of NIA protein levels and post-translational regulation of NIA, whereas the diurnal changes of NIAtranscript levels were not marked-ly modified. Comparison of the in vivochanges in NIA protein and activation and the endogenous changes in glutamine as well as the effect of feeding exogenous glutamine provide indirect evidence that glutamine or related metabolites exert feed-back regulation on the activation and stability of NIA protein.
30 Geiger M, Walch-Piu L, Harnecker J, Schulze E-D, Ludewig F,
• Sonnewald U, Scheible W-R, Stitt M: Enhanced carbon dioxide leads to a modified diurnal rhythm of nitrate reductase activity in older plants, and a large stimulation of nitrate reductase activity and higher levels of amino acids in higher plants.Plant Cell Environ1998,21:253-268.
NIAtranscript typically declines dramatically during the photoperiod, even when the leaves still retain a considerable nitrate pool. One finding in this article is that re-irrigation with a high nitrate concentration late in the pho-toperiod partially reverses this inhibition, indicating that the influx rather than the total pool of nitrate is critical for the regulation of NIAexpression. 31 Vincentz M, Moureaux T, Leydecker MT, Vaucheret H, Caboche M:
Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolialeaves by nitrogen and carbon metabolites.Plant J 1993, 3:313-324.
32 Sivaskar S, Rothstein S, Oaks A: Regulation of the accumulation
• and reduction of nitrate by nitrogen and carbon metabolites in maize seedlings.Plant Physiol1997, 114:583-589.
In roots of maize seedlings, NIAtranscript and NIA activity are increased by sugars and reduced by amino acids including glutamine and asparagine. 33 Tillard P, Passama L, Gojon A: Are amino acids involved in the
• shoot to root control of nitrate uptake in Ricinus communis
plants?J Exp Bot 1998, 49:1371-1379.
It has been widely assumed that amino acids moving in the phloem from the shoot to the roots are responsible for an inhibition of nitrate uptake. This arti-cle shows, using a split-root system, that addition of nitrate to one root sec-tor leads to inhibition of nitrate uptake in the other root secsec-tor, even though the levels of amino acids in the low-nitrogen sector do not increase. 34 Dzuibany C, Haupt S, Fock H, Biehler K, Migge K, Becker TW:
•• Regulation of nitrate reductase transcript levels by glutamine accumulating in the leaves of a ferredoxin-dependent glutamate synthase mutant of Arabidopsis thaliana, and by glutamine provided to the roots.Planta1998, 206:515-522.
It has been frequently proposed, on the basis of correlative studies and the effects of inhibitors, that glutamine, or a closely related metabolite, exerts negative feed-back regulation on NIAexpression. In this study, Arabidopsis
mutants deficient in Fd-GOGAT were grown in the presence of 2% C02to
suppress photorespiration, and then shifted to air levels of CO2. Despite a
large accumulation of glutamine, NIAtranscript responded as in wild-type plants. The authors conclude that glutamine does not exert feed-back regu-lation on NIA, and argue that the repression reported in earlier studies after adding exogenous glutamine may be an indirect effect, due to glutamine inhibiting nitrate uptake.
35 Koch KE: Carbohydrate-modulated gene expression in plants.
Annu Rev Plant Mol Biol Plant Physiol1996, 47:509-540. 36 Jang JC, Sheen J: Sugar sensing in higher plants.Trends Plant Sci
1997, 2:208-214.
37 Morcuende R, Krapp A, Hurry V, Stitt M: Sucrose feeding leads to increased rates of nitrate assimilation, increased rates of a -oxoglutarate synthesis, and increased synthesis of a wide spectrum of amino acids in tobacco leaves.Planta1998, 206:394-409.
38 MacKintosh C: Regulation of cytosolic enzymes in primary
• metabolism by reversible protein phosphorylation.Curr Opin Plant Sci1998, 1:224-229.
A provocative review of the role of protein phosphorylation and of 14-3-3 proteins in regulating primary metabolism and proton pumping at the plas-malemma, that highlights possible interactions between carbon metabolism, nitrogen metabolism and pH regulation.
39 Matt P, Schurr U, Krapp A, Stitt M: Growth of tobacco in short day
an over-riding influence on nitrate assimilation were previously unclear. By investigating the effects of an increasingly long dark period in combination with petiole cooling to inhibit phloem export, it is shown that when sugars fall below a critical level (5–10 µmol/g fresh weight) they strongly inhibit NIA expression and activity, over-riding signals derived from nitrate and nitrogen metabolism. Measurements of amino acid levels indicate that sugars also exert a global inhibition on the amino acid biosynthesis pathways. 40 Botrel A, Kaiser W: Nitrate reductase activation status in barley
•• roots in relation to the energy and carbohydrate status.Planta
1997, 201:496-501.
In studies investigating the stimulatory effect of anaerobiosis on post-trans-lational regulation of nitrate reductase, it was shown that the activation is due to acidification, rather than to changes in the energy status. It is also shown that the decline of NIA activity in prolonged darkness can be prevented by sugar addition.
41 Brouwer R:Nutrient influences on the distribution of the dry matter in the plant.Netherl J Agric Sci1962, 10:399-408. 42 Grime JP, Campbell BD, Mackey JML, Crick JC: Root plasticity,
nitrogen capture and competitive ability. In Plant Root Growth: an Ecological Perspective. Edited by Atkinson D. Oxford: Blackwell Scientific Publications; 1991:381-397.
43 Drew MC, Saker LR: Nutrient supply and the growth of the seminal root system in barley II. Localised compensatory changes in lateral root growth and the rates of nitrate uptake when nitrate is restricted to only one part of the root system.J Exp Bot 1976, 26:79-90. 44 Zhang H, Forde BG:An ArabidopsisMADS-box gene controlling
•• nutrient-induced changes in root architecture.Science1998, 279:407-409.
A MADS-box transcription factor ANR1 was identified in a screen for nitrate-induced genes, and its function investigated in antisense Arabidopsis trans-formants. The overall frequency of lateral root formation in wild-type
Arabidopsis(see also [6··] and [8·]) decreases when plants are grown in high nitrate, whereas local application of nitrate leads to increased lateral root proliferation at the site of application. Antisense inhibition of ANR1
expression increased the sensitivity with which high nitrate inhibited lateral root development, and decreased the effectiveness with which localised applications increased lateral root development. It is also shown that local application of nitrate still induces lateral root formation in severely NIA-defi-cient mutants, showing that nitrate acts as a signal rather than via metabo-lism to promote growth via increased local availability of amino acids. 45 Zhang H, Jennins A, Barlow P, Forde BG: Dual pathways for
•• regulation of root branching by nitrate.Proc Natl Acad Sci USA
1999, in press.
The mechanisms whereby nitrate modulates lateral root formation are further investigated. High nitrate does not inhibit initiation but, rather, acts at the stage where they emerge from the root, apparently delaying final activation of the meristem. This inhibition is accentuated in low-NIA mutants, indicating that tissue nitrate levels are involved in generating the signal.
46 Sagan M, Duc G: Sym28and Sym29, two new genes involved in regulation of nodulation in pea.Symbiosis1996,20:229-245. 47 Novak K, Skrdleta V, Kropacova M, Lisa L, Nemcova M: Interaction of
two genes controlling nodule number in pea.Symbiosis1997, 23:43-62.
48 Bacanamwo M, Harper JE: The feed-back mechanism of nitrate inhibition of nitrogenase activity in soybean may involve asparagine and/or products of its metabolism.Physiol Plantarum
1997, 100:371-377.
49 Markwei CM, LaRue TA: Phenotypic characterisation of sym21, a gene conditioning shoot-controlled inhibition of nodulation in
Pisum sativumcv. Sparkle.Physiol Plantarum1997, 100:927-932. 50 Miller AJ, Smith SJ:Nitrate transport and compartmentation in
cereal root cells.J Exp Bot 199647:843-854.
51 Van de Leij M, Smith S, Miller AJ: Remobilisation of vacuolar stored
• nitrate in barley root cells.Planta1998, 205:64-72.
This paper shows that nitrate accumulated in the vacuole is remobilised after removal of nitrate from the external nutrient medium, and that this remobili-sation occurs without a significant decrease of the cytosolic nitrate concen-tration or NIA activity. As a result, the nitrate concenconcen-tration in the xylem only decreases gradually after depletion of external nitrate.
52 Unden G, Bongaerts J: Alternative respiratory pathways of
Escherichia coli: energetics and transcriptional regulation in response to electron acceptors.Biochim Biophys Acta1997, 1320:217-234.
53 Chiang RC, Cavicchioli R, Gunsalis RP: Locked-on and locked-off signal transduction mutations in the periplasmic domain of the
Escherichia coliNARQ and NARX sensors affect nitrate and nitrate dependent regulation by NARL and NARP.Mol Microbiol
1997, 24:1049-1060.
54 Kim ICH, Kwak JS, Kang JS, Park SC: Transcriptional control of the
glnD gene is not dependent on nitrogen availability in Eschericia coli.Molec Cells1998, 8:483-490.
55 Kamberov ES, Atkinson MR, Ninfa AJ: The Escherichia coliPII signal transduction protein is activated upon binding 2-ketoglutarate and ATP.J Biol Chem 1995, 270:17797-17807. 56 Jiang P, Peliska JA, Ninfa AJ: Enzymological characterisation of the
signal-transducing uridyltransferase/uridyl-removing enzyme of
Eschericia coli and its interaction with the PII protein.Biochemistry
1998, 37:12782-12794.
57 Atkinson MR, Ninfa AJ: Role of the GlnK singnal transduction protein in the regulation of nitrogen assimilation in Eschericia coli.
Mol Microbiol1998, 29:431-477.
58 Xu YB, Cheah E, Carr DD, van Heeswijk WC, Westerhoff HV, Vasudevan SG, Ollis DL: GlnK, a P-II homologue: structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition.J Mol Biol1998, 282:149-165.
59 Garcia Dominguez M, Florencia FJ: Nitrogen availability and electron transport control the expression of glnB gene encoding PII protein in the cyanobacterium Synechocystis sp. PCC 6803.Plant Mol Biol1997, 35:723-734.
60 Lee HM, Flores E, Herrero A, Hoummard J, de Marsac NT: A role for the signal transduction protein in the control of nitrate/nitrite uptake in a cyanobacterium.FEBS Letts1998, 427:291-295. 61 Hsieh M-H, Lam H-M, van de Loo FJ, Coruzzi G: A PII-like protein in
•• Arabidopsis: putative role in nitrogen sensing. Proc Natl Acad Sci USA1998, 95:13965-13970.
This is the first report of a PII protein in plants. It was identified from a EST in Arabidopsis, and is shown to be a nuclear encoded protein whose sequence indicates a plastid localisation. It differs from the classical E. coli
PII system but resembles several recently reported PII homologs in E. coli
and other organisms in showing transcriptional regulation in response to sugars and nitrogen metabolites (see [57–59,60·]) rather than being regu-lated post-translationally via uridenylation/de-uridenylation (see [54–56]). 62 Crawford NM, Arst HN Jr:The molecular genetics of nitrate
assimilation in fungi and plants.Annu Rev Genetics1993, 27:115-146.
63 Marzluf GA: Genetic regulation of nitrogen metabolism in fungi.
Microbiol Mol Biol Reviews1993, 61:17-35.
64 Strauss J, MuroPastor MI, Scazzochhio C: The regulator of nitrate assimilation in ascomycetes is a dimer which binds a
nonrepeated asymmetrical sequence.Mol Cell Biol1998, 18:1339-1348.
65 Quesada A, Hidalgo J, Fernandez E: Three Nrt genes are differentially regulated in Chlamydomonas.Mol Gen Genet1998, 258:373-377. 66 Daniel-Vedele F, Caboche M: A tobacco cDNA clone encoding a
GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi. Mol Genet Gen 1993, 240:365-373. 67 Rastogi R, Bate NJ, Sivanskar S, Rothstein SJ: Footprinting of the
•• spinach nitrite reductase gene promotor reveals the preservation of nitrate regulatory elements between fungi and higher plants.
Plant Mol Biol1997, 34:465-476.
Following earlier investigations showing that a 130 bp upstream region of the spinach nii promotor suffices to confer nitrate-induction of gus expres-sion, a 50 bp region was identified by deletion studies that contained two adjacent gata elements, and bound in vitroa fusion protein containing the zinc finger domain of neurospora crassanit2. These results indicate that nii
expression is mediated by nitrate-specific binding of trans-acting factors to sequences that are preserved between fungi and plants.
68 Truong HN, Caboche M, Daniel-Vedele F: Sequence and
•• characterization of two Arabidopsis thaliana cDNAs isolated by functional complementation of a yeast gln3 gdh1mutant. FEBS Lett1997, 410:213-218.
Two Arabidopsis cDNA sequences were identified that are able to function-ally complement a yeast gln3mutant. GLN3 is related to the positive control genes that mediate repression of nitrate assimilation when preferred nitro-gen sources like ammonium or glutamine are available —AREA and NIT2 in
Aspergillus nidulansand Neurospora crassa, respectively. The Arabidopsis
genes (termed RGA1 and RGA2, restore growth on ammonium) are mem-bers of a multigene family whose other memmem-bers include SCARECROW
that regulates pattern formation in roots. They are identical (see [69]) to GAI
69 Daniel-Vedele F, Filleur S, Caboche M:Nitrate transport: a key step in nitrate assimilation.Curr Opin Plant Biol1998, 1:235-239.
70 Navarro MT, Prieto R, Fernandez E, Galvan A: Constitutive expression of nitrate reductase changes the regulation of nitrate and nitrate transporters in Chlamydomonas reinhardtii.Plant J
1996, 9:819-827.
71 Prieto R, Dubus A, Galvan A, Fernandez E:Isolation and characterisation of two new negative regulatory mutants for nitrate assimilation in Chlamydomonasobtained by insertional mutagenesis.Mol Gen Genet1996, 251:461-471.
72 Murry LE, Rowley N, Dawes IW, Johnston GC, Singer RA: A yeast
• glutamine tRNA signals nitrogen status for regulation of dimorphic growth and sporulation.Proc Natl Acad Sci USA1998, 95:8619-8624.
This intriguing article reports that a glutamine tRNA isoform is involved in nitrogen-signalling in yeast. Mutants lacking this isoform impair nitrogen sensing without impairing protein synthesis, leading to pseudohyphal growth, sporulation even in the presence of preferred nitrogen sources. 73 Kobayashi M, Rodriguez R, Lara C, Omata T: Involvement of the
C-terminal domain of an ATP-binding subunit in the regulation of the ABC-type nitrate/nitrite transporter of the cyanobacterium
Synecoccussp. Strain PCC 7942. J Biol Chem1997, 272:27197-27201.
74. Lorenz MC, Heitman J: The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae. EMBO J 1998, 17:1236-1247.
75. Iraqui J, Vissers S, Bernard F, De Craene J-O, Boles E, Urrestarazu A, André B: Amino acid signaling in Saccharomyces cerevisiae: a
permease-like sensor of external amino acids and the F-box protein Grr1p are required for transcriptional induction of the
AGP1 gene encoding a broad-specificity amino acid permease.
Mol Cell Biol1999, 19:989-1001.
76. Didion T, Regenberg B, Jorgensen MU, Kielland-Brandt MC, Andersen HA: The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in
Saccharomyces cerevisiae. Mol Microbiol1998, 27:643-650. 77 Sakakibara H, Suzuki M, Takei M, Deji A, Taniguchi M, Sugiyama T: A
•• response-regulator homolog possibly involved in nitrogen signal transduction mediated by cytokinin in maize.Plant J1998, 14:337-344.
Addition of nitrate or ammonium to the roots of intact maize plants leads to induction of PPCin the leaves, whereas addition of nitrogen sources to the leaves does not. This paper implicates the synthesis of cytokinins in the roots and cytokinin-induction of a response-regulator homolog ZmCIP1in the leaf signalling pathway in the signalling pathway.
78 Chandok MR, Sopory SK: ZmcPKC70, a protein kinase-type enzyme from maize- biochemical characterisation, regulation by phorbol 12-myristate 13-acetate and its possible involvement in nitrate reductase gene expression.J Biol Chem 1998,
273:19235-19242.
79 Taniguchi M, Kiba T, Sakakibara H, Ueguchi C, Mizuno T, Sugiyama T:
•• Expression of Arabidopsis response regulator homologs is induced by cytokinins and nitrate. FEBS Lett1998,429:259-262. In a sequel to [74], five response regulator homologs are identified in
