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Plant Physiol, January 2001, Vol. 125, pp. 61-64
Nitrogen and Carbon Nutrient and Metabolite Signaling in
Plants
Gloria
Coruzzi* and
Daniel R.
Bush
New York University, Department of Biology, 100 Washington Square
East, New York, New York 10003 (G.C.); and United States Department of
Agriculture-Agricultural Research Service and Department of Plant
Biology, 190 E.R. Madigan Laboratories, University of Illinois,
Urbana, Illinois 61801 (D.R.B.)
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INTRODUCTION |
The effect of nutrients on plant
growth and development has been studied for over 350 years since the
experiments of van Helmont in 1648 (6). Recent studies on nutrient
effects in plants have involved separating their role as building
blocks of organic matter or cofactors from their potential role as
signaling molecules. The first notion that soil nutrients or the
lack thereofcould be sensed by plants was published in
1906 by Brezeale who documented increased transport of
nutrients in response to starvation (9). Fast forward nearly 100 years and transporters for most macronutrients and micronutrients
have been cloned using a variety of techniques including
microbial complementation, expressed sequence tags, and
Arabidopsis mutant isolation. Studies of these cloned transporters have
begun to define the molecular basis for the regulation of uptake
for many of the macronutrients
(NH4+,
NO3 , K+,
Ca2+,
H2PO4,
SO42, and
Mg2+) and micronutrients
(Cl, Zn, Mn2+,
Fe3+, and Cu2+). In a
number of cases, nutrient availability has been shown to affect the
transcription of the transporter gene. A current challenge is to
determine if the nutrient (or lack thereof) is sensed or if the signal
is a derived metabolite. If one considers how long it has taken
researchers to unravel signal transduction pathways for which the
signal is known (e.g. hormones, light), uncovering the workings of
nutrient/metabolic signaling systems is likely to provide challenges to
new researchers well into the new millennium. Even in cases where a
signaling metabolite has been defined, such as for Glc in yeast and in
plants (10,19), the signaling cascades are proving to be remarkably
complex. For example, Glc signaling in plants has been shown to involve
complex cross-talk with hormone signaling pathways (27). Although there is ample evidence for nutrient signaling in relation to a number of
macronutrients and micronutrients, we have focused this historical note
on N and C nutrient and metabolite sensing in plants. We have attempted
to cite early studies that suggested that N or C nutrients, or their
derived metabolites, are sensed and to highlight examples where recent
molecular-genetic studies have begun to identify components of these
sensing/signaling pathways.
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N NUTRIENTS AND METABOLITES AS SIGNALS |
The beneficial effects of saltpeter (KNO3) on plant growth has
been known since the mid-sixteenth century (9). Since that time,
evidence has mounted to support a model in which nitrate acts as a
positive signal necessary for the induction of nitrate uptake and its
reduction (7). In contrast, the metabolized product(s) of nitrate,
ammonium, and its assimilation products Glu and Gln are believed to
exert negative effects on nitrate uptake and reduction. Evidence to
support this model was first discerned using genetic and molecular
approaches in the fungi Neurospora and
Aspergillus (3). Molecular-genetic studies in plants have
also lent support for nitrate and its downstream metabolites to act as
signaling molecules in higher plants (21). However, the role of nitrate as an inducer and ammonium or Gln/Glu as repressor signals is too
simplistic a model. For example, nitrate serves to repress the
expression of a key enzyme in the starch biosynthesis pathway, presumably to divert C skeletons toward the N-assimilatory pathway (18). Below is a brief review of studies that have provided evidence that nitrate, ammonium, or amino acids may serve as signaling molecules
in plants.
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NITRATE AS A SIGNAL |
In the 1950s, physiological studies showed that nitrate treatment
could induce nitrate reductase (NR) activity and nitrate transport,
suggesting a role for nitrate (or nitrite) as a signaling molecule. In
the 1970s, antibodies were used to show that NR was induced by nitrate.
Finally in the 1980s, the gene cloning decade, a molecular basis for
this nitrate regulation began to emerge. Nitrate applications were
shown to induce the accumulation of mRNAs for NR and for a host of
other C- and N-metabolic genes (e.g. SPS, GS, etc.; 3,4). The 1990s saw
the cloning of NRT1 (CHL1) and NRT2 nitrate transporter genes, and it
was shown that nitrate could induce the expression of both (7, 17, 22).
Despite all these studies, it was still not clear how this signaling
pathway worked or whether nitrate or a downstream metabolite was
sensed. In 1997, NR mutants of tobacco that contain very low levels of
NR activity were used to demonstrate that nitrate was capable of
altering gene expression even though it could not be reduced or further metabolized (18). These studies also suggested that nitrate signaling can interact with signals generated further downstream in N metabolism. This was deduced because nitrate treatment of wild-type plants led to
transient induction of genes involved in nitrate uptake and metabolism,
whereas nitrate induction in the NR mutants was sustained. These
findings suggested that downstream products of nitrate reduction
(ammonium or Gln) might act as signaling molecules (see below). A
significant regulatory gene, ANR1, was isolated while searching for
nitrate-induced clones. Repression of ANR1 in antisense transgenics was
shown to impair systemic nitrate repression of lateral root growth and
localized nitrate stimulation of lateral root growth (26). The
isolation on ANR1 was significant, as it succeeded to combine a
nutrient (nitrate) signaling property with a morphological response.
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AMMONIUM AND DERIVED AMINO ACIDS AS SIGNALS |
By contrast to nitrate, evidence for a role of ammonia or a
derived amino acid as a signaling molecule is still rather poor. The
first evidence that ammonium or a derived metabolite could serve as
signals in plants came from studies on the regulation of ammonium
transport. In 1953, N starvation was shown to induce ammonium uptake in
N-starved Chlorella, and similar studies were conducted in
wheat in 1962 (9). In the ensuing decades, ammonium and/or its
assimilation products (Glu/Gln) have each been implicated as negative
regulators of nitrate and ammonium uptake in plants (5,7). A molecular
basis for this regulation came with the cloning of ammonium transporter
(AMT) genes, where it was shown ammonium treatment repressed AMT1
expression (24). More recently, evidence has been presented that Gln, a
product of ammonia assimilation, may effect the repression of
AMT1-mediated ammonium transport in Arabidopsis roots (16). These
studies support the notion that N-assimilation products (Gln or Glu)
might act as signals whose levels are sensed as an indicator for a high
internal N status. Along these lines, putative sensors of Glu in
plants, Glu receptor genes, have been identified in Arabidopsis (14).
Further identification of components of putative amino acid sensing
systems will profit from both forward and reverse genetic approaches.
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C:N SENSING IN PLANTS |
N and C metabolism are tightly linked in almost every biochemical
pathway in the plant. As such, it is not surprising that C metabolites
regulate genes involved in N acquisition and metabolism. Early studies
on NR in 1976, showed that NR activity could be affected by Glc/Suc
(3,4). Those observations were supported by later experiments that
showed sugars induce NR mRNA in dark-adapted, green seedlings (1). The
notion that C and N may have antagonistic relationships as signaling
molecules was reported by Vincentz et al., who showed that light
induction of NR activity and mRNA levels could be mimicked by C
metabolites and that N-metabolites caused repression of NR induction in
tobacco (23). Despite the fact that gene regulation by C:N status has
been demonstrated for a number of N-metabolic genes (21), the
mechanistic basis for this regulation remains to be revealed.
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FUTURE PROSPECTS FOR THE IDENTIFICATION OF N-SIGNALING
COMPONENTS |
The isolation of mutants defective in components of the N or C:N
signaling pathways as well as the use of insertion mutants in
transporter genes are very promising tools to identify sensing mechanisms in the future. The success of these approaches will very
much depend on the development of adequate screening tests to make a
sensing response visible or measurable. The completion of the
Arabidopsis genome sequencing project and the development of genomic
technologies has aided in the identification of components in
N-signaling systems. For example, in 2000 Wang et al. used micro-array
analysis to identify the induction by nitrate of putative regulatory
genes (e.g. MYB transcription factors, protein kinases, etc.),
metabolic enzymes, and novel gene products (25).
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C METABOLITES AS SIGNALS |
Early evidence of C metabolite signaling was provided in the 1960s
by experiments linking the rate of photosynthesis with assimilate
partitioning (for review, see 15). These initial observations were
followed up with many experiments in the 1970s that examined the
relationship between photosynthetic capacity in source tissue with
carbohydrate use in sinks (for review, see 8). Gifford and Evans'
analysis of those results led to the conclusion that sink tissue plays
a pivotal role in controlling assimilate partitioning. However, despite
this conclusion, there were many contradictory observations in the
literature and the identity of a potential integrating signal (such as
turgor, a metabolite or plant growth hormone) was not known.
Moreover, multiple examples of allosteric regulation of enzyme
activities by key metabolites added another layer of complexity to
understanding assimilate partitioning as a globally integrated system.
The first evidence that C metabolites play a direct role in regulating
photosynthetic activity at the transcriptional level was Sheen's
demonstration that sugars down-regulate photosynthetic gene expression
in protoplasts isolated from maize mesophyll (19). In those
experiments, seven photosynthetic genes were coordinately repressed by
Glc and Suc. Subsequent experiments with a variety of sugar analogs
suggested hexokinase plays a key role in transducing a hexose-dependent
signal in the maize protoplast system and that conclusion was then
supported with genetic results using hexokinase antisense plants (10).
Although earlier publications provided evidence of links between
carbohydrate status and changes in growth or gene expression, Sheen's
results were the first to demonstrate a direct effect of C metabolites
on the expression of photosynthetic genes. Evidence that these
observations parallel changes in photosynthetic activity in intact leaf
tissue was shown in leaf girdling experiments that documented
carbohydrate-dependent decreases in photosynthetic gene expression and
the rate of photosynthesis (12).
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THERE ARE MULTIPLE PATHWAYS OF C-METABOLITE REGULATION |
Carbohydrate-mediated changes in gene expression have been
demonstrated for a variety of physiological and developmental processes (11,20). In general, these responses fall into two broad categories: (a) those that increase gene expression under carbohydrate rich conditions and (b) those that increase expression in depleted conditions (Dr. Koch's "feast and famine" responses, 11).
Carbohydrate-modulated response pathways are also differentiated by
tissue and developmental specificity and the identity of the signaling
molecule. For example, evidence for Suc-mediated changes in gene
expression, not mimicked by hexoses, has been demonstrated for a Suc
symporter in sugar beet leaf tissue whose message and transport
activity are down-regulated when Suc accumulates in the leaf (2). Even
hexose signaling may be transduced by a hexokinase-independent system
linked to a membrane bound hexose transporter (11,20).
| |
METABOLITE REGULATION OF GENE EXPRESSION IS ONE COMPONENT OF A WEB
OF INTERLINKED REGULATORY NETWORKS |
In addition to the C:N cross-talk discussed above, genetic
analysis of C-metabolite signaling has recently shown that it is also
linked to ethylene (27) and abscisic acid response pathways (13).
Genetic analysis of gin1, a Glc-insensitive mutant in Arabidopsis, showed that this gene product acts downstream of the hexokinase in the
Glc signaling pathway. Significantly, gin1 insensitivity to
Glc repression of cotyledon and shoot development was phenocopied by
ethylene precursor treatment of wild-type plants and etr1-1, an
ethylene insensitive mutant, was shown to be Glc hypersensitive. Epistasis analysis placed GIN1 downstream of the ethylene
receptor, ETR1. Taken together, these results suggest GIN1
may integrate antagonistic signals from these two pathways in
controlling plant development (27). Analysis of additional
Glc-insensitive mutants has also identified cross-talk with abscisic
acid signaling (13) and, based on paclobutrazol-insensitive seed
germination, gibberellin (S. Gibson, personal communication). Given the
central role of C metabolism in plant cell biology, additional examples
of regulatory cross-talk are likely to be uncovered.
Our understanding of metabolite regulation of gene expression has grown
by quantum leaps over the last 10 years. As with many areas of
biological inquiry, many of these advances can be attributed to the
development of genetics and recombinant DNA technology as potent tools
in physiological research. We are on the threshold of the next
revolution in biology as genomics leads us into a new century. The
emerging tools of genomics and bio-informatics will allow us to
identify the interacting pathways that control gene expression in
response to changes in N and C status, which should give us new insight
into how plants modulate growth and development in response to
fluctuating environmental conditions.
| |
ACKNOWLEDGMENTS |
We would like to thank Nigel Crawford, Anthony Glass, and Nico
Von Wiren for their guidance in documenting the history of N
sensing/signaling in plants, and Sue Gibson for sharing results prior
to publication.
| |
FOOTNOTES |
*
Corresponding author; e-mail gloria.coruzzi{at}nyu.edu; fax
212-995-4204.
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TOP
INTRODUCTION
N NUTRIENTS AND METABOLITES...