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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
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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.