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178 Nitrate regulation of metabolism and growth Mark Stitt 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: [email protected] Current Opinion in Plant Biology 1999, 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 metabolites formed during nitrate assimilation, or, more indirectly, as a result of changes in other cell constituents or the overall rate of growth. In addition, signals originating from downstream events may exert negative feed-back regulation on earlier steps in nitrate acquisition. In a given situation, it is likely that several signals interact to condition 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) activity [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 independently 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 summarises 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-oxoglutarate [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 utilisation requires continued synthesis of sucrose. These results open up new strategies to engineer carbon partitioning to starch accumulation. Nitrate regulation of metabolism and growth Stitt 179 Table 1 Genes known to be regulated by nitrate-mediated signalling. Physiological function Gene Protein Regulated in root References Nitrate uptake NRT1 NRT2 high affinity NO3 transporter low affinity NO3 transporter + + [13•,14,15••,21] [14,15••,22,23] NIA NII nitrate reductase nitrite reductase + + + + [7••,11,12,16••,18] [7••,11,16••] Ammonium assimilation GLN1 GLN2 GLU plastid glutamine synthetase cytosolic glutamine synthetase GOGAT + + + + + + [7••,17] [7••,17,24•] [7••] Organic acid metabolism PPC PKc CS ICDH1 phosphoenolpyruvate carboxylase cytosolic pyruvate kinase citrate synthase NADP-isocitrate dehydrogenase + + + + + + + + [7••] [7••] [7••] [7••] Redox metabolism PGD FNR FD 6-phosphogluconate dehydrogenase ferredoxin:NADP oxidoreductase ferredoxin ? ? ? + + + [20••] [19] [26••] Starch synthesis AGPS ADP-glucose pyrophosphorylase (regulatory subunit) – ? [7••] Nitrate assimilation Regulated in leaf Nitrate and nitrite reduction consume NADH in the cytoplasm and reduced ferredoxin in the plastid, respectively. Reducing equivalents can be provided directly or indirectly 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••], ferredoxin [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 carbon metabolism. This allows a reprogramming of nitrogen 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. NIA NO3 GLN1 GLN2 NII NO2 NH3 Gln PGD FD FNR Redox equivalents GLU Glu Amino acids αOG ICDH Citrate Malate CS AcCoA OA Pyr Starch PPC PK AGPS PEP Sucrose CO2 SPS Current Opinion in Plant Biology 180 Physiology and metabolism 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 nitrogen [7••,27•] and in elevated CO2, where faster growth often leads to a marked decrease of nitrate [27•,28], in particular 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 expression, 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 metabolism. This is illustrated by the three general observations. First, whereas nitrate addition leads to a transient induction of genes involved in nitrate uptake and assimilation 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 corresponding 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-signalling 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 repress NIA in maize roots [32•]. Two recent studies, however, point towards more a complicated 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 exogenous glutamine used in previous experiments acts indirectly, by inhibiting nitrate uptake [34••]. Alternative explanations would be that NIA is repressed by a specific glutamine pool which is not affected in the glu mutants, or that the increase of glutamine is compensated for by changes in other unidentified metabolite pools. Nitrogen metabolism is complicated in leaves of C-3 plants by fluxes of ammonium around the photorespiratory 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 assimilation 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 activation 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 photosynthesis) to α-oxoglutarate [38•]. When sugars are low they exert a strong negative regulation on NIA expression, 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 assimilation leads to alkalisation, and ammonium assimilation leads to acidification. Whereas roots can exchange protons directly or indirectly with the soil, assimilation of nitrate in leaves requires the synthesis and export of malate to the roots, where it is decarboxylated. Intriguingly, alkalisation leads to post-transcriptional inactivation of NIA [40••]. The mechanism involves phosphorylation and binding of 14-3-3 proteins, which is reminiscent of the mechanism for activation of the plasmalemma proton-pumping ATPase [37]. It will be fascinating to learn more about the regulatory interactions between cellular pH, organic acid metabolism, and nitrogen metabolism. Indeed, changes in pH might provide a far more pressing reason for rapid regulation of nitrate and ammonium metabolism than accumulation of ammonium, glutamine and other amino acids, as these can accumulate to relatively high levels in plants without harmful effects. Nitrate regulation of metabolism and growth Stitt 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 forage a larger soil volume for nitrogen-containing nutrients, and direct their root growth in response to the spatial distribution 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 splitroot 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-deficient Arabidopsis is 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 important whole plant processes like flowering and senescence still has to be investigated. It is also intriguing that hypernodulating mutants have been identified where the increased nodule number is due to loss of a shoot-dependent 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 NIA decrease after illumination, and re-fertilisation with high nitrate concentrations later in the light period leads to re-induction of NIA, even 181 Figure 2 NO3 NO3 Local NO3 Current Opinion in Plant Biology 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. 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 mechanism regulates an operon that contains genes for a set of electron transport components and an anaerobiosis-specific NIA which 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 autophosphorylating histidine kinase sensor components (NARX, NARQ) to the corresponding DNA-binding response element (NARL, NARP). Homologous systems occur in higher plants, but it is not known any if any of them represent 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-oxoglutarate are sensed in E. coli via a cascade involving the bifunctional uridyl transferase/uridyl removing enzyme (UT–UR), the regulatory PII protein 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 probably represent the sensor, as they are constitutively expressed [54], and their activity is regulated by metabolites [55,56]. There is recent evidence in E. coli and other species [57,58] including cyanobacteria [59], however, for a 182 Physiology and metabolism PII homolog that is transcriptionally regulated by metabolites. 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••]. GLB1 lacks 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 regulating 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 designated 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 asymmetrical nucleotide sequence [64] to mediate a pathway-specific induction of nitrate uptake and NIT3. NIT2/AREA is member of the GATA family of transcription 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 further 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 GAI and 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 regulation 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 nitrogen source quality in yeast, and regulates the uptake and use of non-preferred nitrogen sources and sporulation independently of changes in the rate of protein synthesis [72•]. Transplant proteins can also act in yeast [73–76]. Recent studies have elucidated novel downstream signalling components in nitrate-signalling 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 lateral 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 regulated. 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 system 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 NADHdependent-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 nitratespecific induction of the nitrate assimilation pathway in leaves may involve calcium and protein phosphorylation [24•] via protein kinase C [78]. Induction of PPC in whole plants, on the other hand, is associated with increased levels 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 PPC and pZmCIP1 transcripts could be mimicked by feeding cytokinins to detached leaves [77••]. pZmCIP1 encodes a nitrogen- and cytokinin-induced homolog of the response element of bacterial two component signalling systems. A small family of response regulator proteins (ARR3–ARR7) showing a similar cytokinin- and nitrate-dependent expression has recently been identified in Arabidopsis [79••]. It will be fascinating to learn which endogenous ligands interact with these cytokinin and nitrogen-induced sensors, and what phenotypes result when their expression is disrupted. Thus, although molecular analysis of nitrate-sensing in plants is in its infancy, there are tantalising hints of interactions with fundamental processes in plant metabolism and development. Whereas nitrate has only been implicated in pathway-specific regulation in bacteria and fungi, it may have a more far-reaching role in higher plants. This may reflect the importance of nitrate as a nitrogen source for many plants and habitats, and inherent differences between plants and microbes including their ability to store large amounts of nitrate, possible complicating effects of photorespiration, and the need to modulate allocation and development in a multi-cellular Nitrate regulation of metabolism and growth Stitt 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 availability of nitrate. We can expect that molecular analysis of nitrate-signalling will provide further important insights into the networking of metabolism, and into the mechanisms whereby plant development is modulated by response to changes in the environment. shoot–root ratio than in nitrogen-replete wild-type plants, even though amino acid and protein levels and overall growth rates resembled those in nitrogendeficient wild-type plants. Split-root experiments showed that root growth was decreased due to accumulation of nitrate in the shoot. High local levels of nitrate in the rooting medium stimulated growth, also via mechanisms that did not require metabolism of nitrate. 7 •• Scheible W-R, Gonzales-Fontes A, Lauerer M, Müller-Röber B, Caboche M, Stitt M: Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell 1997, 9:783-798. Tobacco mutants and transformants with decreased NIA expression were grown on low and high nitrate and investigated with respect to transcripts, enzyme activities and metabolites in nitrogen and carbon metabolism in leaves and roots. Accumulation of nitrate led to increased expression and activity of genes involved in nitrate and ammonium assimilation (NIA, NII, GLN1, GLN2, GLU) and organic acid synthesis (PPC, cytosolic PK, CS, ICDH-1) and accumulation of malate and α-oxoglutarate, and to decreased expression of AGPS and decreased levels of starch. The changes in transcripts were independent of the metabolism of the nitrate, and occurred within 2–4 h after supplying nitrate to the roots of whole plants. 8 • Stitt M, Feil R: Lateral root frequency decreases when nitrate accumulates in tobacco transformants with low nitrate reductase activity: consequences for the regulation of biomass partitioning between shoots and roots. Plant Soil 1999, in press In a sequel to [6••], low-NIA tobacco transformants were grown on nutrient agar at various nitrate supplies in the presence or absence of sucrose. The inhibition of root growth when nitrate accumulates in the plant was accompanied by a marked decrease in the frequency of growing lateral roots, which was not relieved by including sucrose in the medium. 9 Jackson WA, Flesher D, Hageman RH: Nitrate uptake by darkgrown corn seedlings: some characteristics of apparent induction. Plant Physiol 1973, 51:120-127. 10 Shaner DL, Boyer JS: Nitrate reductase activity in maize (Zea mays L.) leaves. I. Regulation by nitrate flux. Plant Physiol 1976, 58:499-504. 11 Vaucheret H, Chabaud M, Kronenberger J, Caboche M: Functional complementation of tobacco and Nicotiana plumbaginifolia nitrate reductase deficient mutants by transformation with the wild-type alleles of the tobacco structural genes. Mol Gen Genet 1990, 220:468-474. 12 Hoff T, Truong H-N, Caboche M: The use of mutants and transgenic plants to study nitrate metabolism. Plant Cell Environ 1994, 17:486-506. 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: 13 • Krapp A, Fraisier V, Scheible W-R, Quesada A, Gojon A, Stitt M, Caboche M, Daniel-Vedele F: Expression studies of Nrt2:1Np, a putative high affinity nitrate transporter: evidence for its role in nitrate uptake. Plant J 1998, 6:723-732. Investigations in wild-type tobacco and low-NIA transformants showed that NRT2:1Np (encoding a high affinity nitrate transporter) is induced by nitrate, and repressed by ammonium or a product of ammonium metabolism. 14 • of special interest •• of outstanding interest 1. Marschner M: Mineral Nutrition of Higher Plants, edn 2. London: Academic Press Ltd; 1995. 2 Crawford NM: Nitrate: nutrient and signal for plant growth. Plant Cell 1995, 7:859-868. 3 von Wiren N, Gazzarrinni S, Frommer WB: Regulation of mineral • nitrogen uptake in plants. Plant Soil 1997, 196:191-199. See annotation for [4•]. 4 • Forde BG, Clarkson DT: Nitrate and ammonium nutrition of plants: physiological and molecular perspectives. Adv Bot Res 1999, in press. Together with [3•], two excellent reviews of the classic studies of the physiology of nutrient uptake, and their relation to the recent advances following the cloning of genes encoding nitrate transport systems. 5 6 •• Bernier G, Havelange A, Houssa C, Petitjean A, Lejeune P: Physiological signals that induce flowering. Plant Cell 1993, 5:1147-1155. Scheible W-R, Lauerer M, Schulze E-D, Caboche M, Stitt M: Accumulation of nitrate in the shoot acts as signal to regulate shoot-root allocation in tobacco. Plant J 1997, 11:671-691. Tobacco mutants and transformants with decreased expression of NIA were grown at various nitrogen supplies, and the levels of nitrate, amino acids, protein, overall growth and shoot–root allocation investigated. Accumulation of nitrate in low-NIA genotypes inhibited root growth, resulting in a higher 183 Filleur S, Danielle-Vedele F: Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display. Planta 1998, 207:461-489. 15 •• Lejay L, Tillard P, Lepetit M, Olive FD, Filleur S, Daniel-Vedele F, Gojon A: Molecular and functional characterisation of two NO3uptake systems by N- and C-status of Arabidopsis plants. Plant J 1999, in press. Transcript levels for NRT2:1 and NRT1 (encoding a high and low affinity nitrate transporter) and nitrate uptake rates were studied in wild-type Arabidopsis and low-NIA mutants growing at various nitrogen supplies. Diurnal changes and the response to added sugar were also analysed. Nitrate induces both transporters, whereas ammonium or products of ammonium assimilation repress both transporters, and sugars lead to increased expression of both transporters. Further, it is shown that the changes in transcript levels correlate with changes in the rates of nitrate uptake. 16 •• Migge A, Carrayol E, Kunz C, Hirel B, Fock H, Becker T: The expression of the tobacco genes encoding plastidic glutamine synthetase or ferredoxin-dependent glutamate synthase does not depend on the rate of nitrate reduction, and is unaffected by suppression of photorespiration. J Exp Bot 1997, 48:1175-1184. Inhibition of nitrate assimilation by addition of tungsten led to increased expression of NIA and NII but did not alter expression of GS2 and GLU, indicating that downstream products of nitrate assimilation exert feed-back control on the nitrate assimilation pathway, but not on the ammonium assimilation pathway. 17 Redinbaugh MG, Campbell WH: Glutamine synthetase and ferredoxin-dependent glutamate synthase expression in the maize (Zea mays) root: primary response to nitrate. Plant Physiol 1993, 101:1249-1255. 184 Physiology and metabolism 18 Gowri G, Ingemarsson B, Redinbaugh MG, Campbell WH: Nitrate reductase transcript is expressed in the primary response of maize to environmental nitrate. Plant Mol Biol 1992, 18:55-64. 19 Ritchie SW, Redinbaugh MG, Shiraishi N, Verba JM, Campbell WH: Identification of a maize root transcript expressed in the primary response to nitrate: characterization of a cDNA with homology to ferredoxin-NADP(+) oxidoreductase. Plant Mol Biol 1994, 26:679-690. 20 •• Redinbaugh MG, Campbell WH: Nitrate regulation of the oxidate pentose phosphate pathway in maize root plastids: induction of 6-phosphogluconate activity, protein and transcript levels. Plant Science 1998, 134:129-140. Addition of nitrate to maize plants leads to a rapid increase of transcript encoding 6-phosphogluconate dehydrogenase via a mechanism that does not require the intervening synthesis of new proteins. Longer treatments lead to an increase of 6-phosphogluconate protein and activity. This is the first report directly linking nitrate to regulation of the oxidative pentose phosphate pathway, which is required to supply reducing equivalents for nitrate assimilation in roots. 21 Amarasingh BHRR, de Bruxelles GL, Braddon M, Onyeocha I, Forde BG, Udvardi MK: Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max). Planta 1998, 206:44-52. 22 Tsay YF, Schroeder JI, Feldmann A, Crawford NM: The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate inducible nitrate transporter. Cell 1993, 72:705-713. 23 Lauter FR, Ninneman O, Bucher M, Riesmeier J, Frommer WB: Preferential expression of an ammonium transporter and of two nitrate transporters in root hairs of tomato. Proc Natl Acad Sci USA 1998, 88:8139-8144. 24 • Sakakibara H, Kobayashi K, Deji A, Sugiyama T: Partial characterisation of the signalling pathway for the nitratedependent expression of genes for nitrogen-assimilatory enzymes using detached maize leaves. Plant Cell Physiol 1997, 38:837-843. Addition of nitrate to maize leaves leads to rapid induction of NIA, NII, plastid GLN2 and GLU, via a mechanism that does not require an intervening synthesis of new proteins. The effects of chelators, calcium analogs and protein kinase and phosphatase inhibitors indicate a role for Ca2+ and protein phosphorylation in the signalling mechanism 25 • Gojon A, Dapoigny L, Lejay L, Tillard P, Rufty TW: Effects of genetic modification of nitrate reductase expression on 15NO3- uptake and reduction in Nicotiana plants. Plant Cell Environ 1998, 21:43-53. 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 represses nitrate uptake, and provide direct evidence that changes 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 Physiol 1997, 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 CO2 on 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 CO2 are 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 Environ 1999, 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 nia genes compensate by modifying the diurnal regulation of transcription, posttranslational 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 NIA transcript levels were not markedly modified. Comparison of the in vivo changes 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 Environ 1998, 21:253-268. NIA transcript 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 photoperiod partially reverses this inhibition, indicating that the influx rather than the total pool of nitrate is critical for the regulation of NIA expression. 31 Vincentz M, Moureaux T, Leydecker MT, Vaucheret H, Caboche M: Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolia leaves 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 Physiol 1997, 114:583-589. In roots of maize seedlings, NIA transcript 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 article shows, using a split-root system, that addition of nitrate to one root sector leads to inhibition of nitrate uptake in the other root sector, 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. Planta 1998, 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 NIA expression. In this study, Arabidopsis mutants deficient in Fd-GOGAT were grown in the presence of 2% C02 to suppress photorespiration, and then shifted to air levels of CO2. Despite a large accumulation of glutamine, NIA transcript responded as in wild-type plants. The authors conclude that glutamine does not exert feed-back regulation 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 Physiol 1996, 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 aoxoglutarate synthesis, and increased synthesis of a wide spectrum of amino acids in tobacco leaves. Planta 1998, 206:394409. 38 • MacKintosh C: Regulation of cytosolic enzymes in primary metabolism by reversible protein phosphorylation. Curr Opin Plant Sci 1998, 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 plasmalemma, 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 conditions leads to high starch, low sugars, altered diurnal changes of the nia transcript and low nitrate reductase activity, and an inhibition of amino acid synthesis. Planta 1998, 207:27-41. It was well established that sugar addition leads to an increase of NIA transcript and activity in detached leaves, but the situations in which sugars exert Nitrate regulation of metabolism and growth Stitt 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-translational 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. 185 Escherichia coli NARQ 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 Cells 1998, 8:483-490. 55 Kamberov ES, Atkinson MR, Ninfa AJ: The Escherichia coli PII signal transduction protein is activated upon binding 2ketoglutarate 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 Microbiol 1998, 29:431-477. 41 Brouwer R: Nutrient influences on the distribution of the dry matter in the plant. Netherl J Agric Sci 1962, 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. 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 Biol 1998, 282:149-165. 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. 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 Biol 1997, 35:723-734. Zhang H, Forde BG: An Arabidopsis MADS-box gene controlling nutrient-induced changes in root architecture. Science 1998, 279:407-409. A MADS-box transcription factor ANR1 was identified in a screen for nitrateinduced genes, and its function investigated in antisense Arabidopsis transformants. 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-deficient mutants, showing that nitrate acts as a signal rather than via metabolism to promote growth via increased local availability of amino acids. 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 Letts 1998, 427:291-295. 44 •• 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. 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 USA 1998, 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 regulated 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 Genetics 1993, 27:115146. 63 Marzluf GA: Genetic regulation of nitrogen metabolism in fungi. Microbiol Mol Biol Reviews 1993, 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 Biol 1998, 18:1339-1348. 45 •• 46 Sagan M, Duc G: Sym28 and Sym29, two new genes involved in regulation of nodulation in pea. Symbiosis 1996, 20:229-245. 65 47 Novak K, Skrdleta V, Kropacova M, Lisa L, Nemcova M: Interaction of two genes controlling nodule number in pea. Symbiosis 1997, 23:43-62. Quesada A, Hidalgo J, Fernandez E: Three Nrt genes are differentially regulated in Chlamydomonas. Mol Gen Genet 1998, 258:373-377. 66 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. 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 •• 48 49 Markwei CM, LaRue TA: Phenotypic characterisation of sym21, a gene conditioning shoot-controlled inhibition of nodulation in Pisum sativum cv. Sparkle. Physiol Plantarum 1997, 100:927-932. 50 Miller AJ, Smith SJ: Nitrate transport and compartmentation in cereal root cells. J Exp Bot 1996 47:843-854. 51 Van de Leij M, Smith S, Miller AJ: Remobilisation of vacuolar stored • nitrate in barley root cells. Planta 1998, 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 remobilisation occurs without a significant decrease of the cytosolic nitrate concentration or NIA activity. As a result, the nitrate concentration 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 Acta 1997, 1320:217-234. 53 Chiang RC, Cavicchioli R, Gunsalis RP: Locked-on and locked-off signal transduction mutations in the periplasmic domain of the 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 Biol 1997, 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 expression, a 50 bp region was identified by deletion studies that contained two adjacent gata elements, and bound in vitro a fusion protein containing the zinc finger domain of neurospora crassa nit2. 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 gdh1 mutant. FEBS Lett 1997, 410:213-218. Two Arabidopsis cDNA sequences were identified that are able to functionally complement a yeast gln3 mutant. GLN3 is related to the positive control genes that mediate repression of nitrate assimilation when preferred nitrogen sources like ammonium or glutamine are available — AREA and NIT2 in Aspergillus nidulans and Neurospora crassa, respectively. The Arabidopsis genes (termed RGA1 and RGA2, restore growth on ammonium) are members of a multigene family whose other members include SCARECROW that regulates pattern formation in roots. They are identical (see [69]) to GAI and RGA, that are involved in gibberellin signal transduction. 186 Physiology and metabolism 69 Daniel-Vedele F, Filleur S, Caboche M: Nitrate transport: a key step in nitrate assimilation. Curr Opin Plant Biol 1998, 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 Chlamydomonas obtained by insertional mutagenesis. Mol Gen Genet 1996, 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 USA 1998, 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 Cterminal domain of an ATP-binding subunit in the regulation of the ABC-type nitrate/nitrite transporter of the cyanobacterium Synecoccus sp. Strain PCC 7942. J Biol Chem 1997, 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 Biol 1999, 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 Microbiol 1998, 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 J 1998, 14:337-344. Addition of nitrate or ammonium to the roots of intact maize plants leads to induction of PPC in 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 ZmCIP1 in the leaf signalling pathway in the signalling pathway. 78 79 •• 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. 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 Lett 1998, 429:259-262. In a sequel to [74], five response regulator homologs are identified in Arabidopsis whose expression is increased by addition of nitrate or cytokinins. The identity of the ligand for the response regulators is still unknown, but they could represent a self-amplification loop or a change in sensitivity to further unidentified signalling components.