Plant, Cell and Environment (1999) 22, 583–621
ORIGINAL ARTICLE OA 220
The interaction between elevated carbon dioxide and nitrogen
nutrition: the physiological and molecular background
M. STITT & A. KRAPP
Botanisches Institut, Im Neuenheimer Feld 360, D-69120, Germany
ABSTRACT
This review first summarizes the numerous studies that
have described the interaction between the nitrogen
supply and the response of photosynthesis, metabolism
and growth to elevated [CO2]. The initial stimulation of
photosynthesis in elevated [CO2] is often followed by a
decline of photosynthesis, that is typically accompanied by
a decrease of ribulose-1,5-bisphosphate carboxylase/oxy-
genase (Rubisco), an accumulation of carbohydrate espe-
cially starch, and a decrease of the nitrogen concentration
in the plant. These changes are particularly marked when
the nitrogen supply is low, whereas when the nitrogen
supply is adequate there is no acclimation of photosynthe-
sis, no major decrease in the internal concentration of
nitrogen or the levels of nitrogen metabolites, and growth
is stimulated markedly. Second, emerging evidence is dis-
cussed that signals derived from nitrate and nitrogen
metabolites such as glutamine act to regulate the expres-
sion of genes involved in nitrate and ammonium uptake
and assimilation, organic acid synthesis and starch accu-
mulation, to modulate the sugar-mediated repression of
the expression of genes involved in photosynthesis, and to
modulate whole plant events including shoot–root alloca-
tion, root architecture and flowering. Third, increased
rates of growth in elevated [CO2] will require higher rates
of inorganic nitrogen uptake and assimilation. Recent
evidence is discussed that an increased supply of sugars
can increase the rates of nitrate and ammonium uptake
and assimilation, the synthesis of organic acid acceptors,
and the synthesis of amino acids. Fourth, interpretation of
experiments in elevated [CO2] requires that the nitrogen
status of the plants is monitored. The suitability of differ-
ent criteria to assess the plant nitrogen status is critically
discussed. Finally the review returns to experiments with
elevated [CO2] and discusses the following topics: is, and if
so how, are nitrate and ammonium uptake and
metabolism stimulated in elevated [CO2], and does the
result depend on the nitrogen supply? Is acclimation of
photosynthesis the result of sugar-mediated repression of
gene expression, end-product feedback of photosynthesis,
nitrogen-induced senescence, or ontogenetic drift? Is the
Correspondence: Mark Stitt. Fax: +49–6221–54–5859; e-mail:
mstitt@botanik1.bot.uni-heidelberg.de
© 1999 Blackwell Science Ltd
EN
accumulation of starch a passive response to increased
carbohydrate formation, or is it triggered by changes in
the nutrient status? How do changes in sugar production
and inorganic nitrogen assimilation interact in different
conditions and at different stages of the life history to
determine the response of whole plant growth and alloca-
tion to elevated [CO2]?
Key-words: elevated carbon dioxide; nitrate; nitrogen; nitrogen
signalling; photosynthesis; Rubisco; starch; sugar signalling.
Abbreviations: AGPase, ADP glucose pyrophosphorylase;
GS, glutamine synthetase; GOGAT, glutamate : oxoglutarate
amino transferase; NADP-ICDH, NADP-dependent isocitrate
dehydrogenase; NR, nitrate reductase; OPPP, oxidative pen-
tose phosphate pathway; 3PGA, glycerate-3-phosphate;
PEPCase, phosphoenolpyruvate carboxylase; Rubisco, ribu-
lose-1,5-bisphosphate carboxylase/oxygenase; SPS, sucrose
phosphate-synthase.
1 INTRODUCTION
Elevated [CO2] leads to increased rates of carboxylation
and decreased rates of oxygenation of ribulose-1,5-bispho-
sphate carboxylase/oxygenase (Rubisco) in C3 plants,
resulting in a higher net rate of photosynthesis and
increased synthesis of carbohydrate (Stitt 1991; Bowes
1996; Drake, Gonzàlez-Meler & Long 1997). Viewed in a
broader context, however, carbon dioxide is just one of
many inorganic substrates that are required by plants, and
the long-term response of photosynthesis and growth to
elevated [CO2] will depend, for two basic reasons, on the
availability of mineral nutrients and the way in which they
are utilized by the plant.
Firstly, the higher rates of growth in elevated [CO2]
will lead to an increased demand for mineral nutrients.
This could be met by using nutrients more efficiently
and/or by increasing the rate at which minerals are
absorbed and assimilated by the plant. To understand the
response of plant metabolism and growth to elevated
[CO2] we will therefore need to consider how mineral
nutrients are acquired and used in the plant. It can be
expected that the response will vary, depending on the
species involved, the level of nutrition the plant is
receiving, and the mineral in question.
583
584 M. Stitt and A. Krapp
Secondly, the acceleration of growth and increase of
biomass in elevated [CO2] may change the nutrient status
in the plant. This is a very real possibility, unless the nutri-
ent supply is super-optimal or is increased in parallel with
the rate of plant growth. Although changes in the nutri-
tional status will not invalidate field studies whose aim is
to describe the response of natural populations to elevated
[CO2], it will be important to recognize if and when the
nutrient state is being modified in order to interpret the
results and relate them to studies at other locations and on
other species. Changes in the nutritional status will have a
serious consequence for studies in controlled conditions
which are analysing the physiological and molecular
mechanisms whereby elevated [CO2] affects plant
metabolism and growth, because failure to recognize a
change in the nutritional status could lead to misinterpreta-
tion of the results.
The following review will concentrate on the interaction
between elevated [CO2] and nitrogen nutrition, because
this interaction has been more intensively investigated than
the interaction with other nutrients, and because the back-
ground knowledge about nitrogen uptake, assimilation and
utilization is more extensive than for other nutrients. We (i)
summarize the results from numerous descriptive studies
that have investigated the interaction between elevated
[CO2] and nitrogen nutrition. We then discuss the possible
physiological and molecular background to this interac-
tion, in particular (ii) how metabolism, allocation and
growth are regulated in response to changes in the nitrogen
status of the plant and (iii) whether mechanisms are known
that could stimulate the uptake and assimilation of inor-
ganic nitrogen or increase the nitrogen use efficiency in
response to the conditions pertaining in elevated [CO2],
especially an increased rate of carbohydrate formation. We
then consider (iv) what is the best way to monitor the nitro-
gen status of a plant before (v) returning to the studies of
plant metabolism and growth in elevated [CO2] and con-
sidering how they can be interpreted in terms of our back-
ground knowledge about the interactions between carbon
and nitrogen metabolism and signalling processes, and to
what extent the interpretation may be complicated or
falsified by changes in the nitrogen status.
2 THE PHENOMENOLOGY OF THE
NITROGEN-ELEVATED CARBON DIOXIDE
INTERACTION
2.1 Acclimation of photosynthesis is more
marked in nitrogen-limited plants
Although the initial stimulation of net photosynthesis is
sometimes retained during long-term exposure to elevated
[CO2] (Idso & Kimball 1992; Gunderson, Norby &
Wullschleger 1993; Teskey 1995), it is often partially
reversed or abolished (Stitt 1991; Oechel et al. 1993; Sage
1994; Nie et al. 1995a; Jacob, Greitner & Drake 1995;
Tissue, Thomas & Strain 1997; Drake et al. 1997) in a
process termed the ‘acclimation’ of photosynthesis to
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
elevated [CO2]. Acclimation is usually accompanied by
alterations in the gas exchange characteristics that are
indicative of a decreased carboxylation capacity (Stitt 1991;
Sage 1994; but see also Section 5.2.1) and decrease of
Rubisco activity or Rubisco protein in many species (Drake
et al. 1997), including tobacco (Sicher & Kremer 1994;
Sicher, Kremer & Rodermel 1994), tomato (Van Oosten,
Wilkins & Beresford 1994), Scirpus olneyi (Jacob et al.
1995), wheat (Nie et al. 1995a; Rogers et al. 1996; Sicher &
Bunce 1997), barley (Sicher & Bunce 1997), rice (Vu et al.
1997), Arabidopsis thaliana (Cheng, Moore & Seeman
1998), Panicum laxum (von Ghannoum et al. 1997),
loblolly pine (Tissue, Thomas & Strain 1993; Tissue et al.
(1997), cherry (Atkinson et al. (1997), oak (George, Gerant
& Dizengremel 1996) and cork oak (Faria et al. 1996).
Prolonged exposure to elevated [CO2] has also been shown
to lead to a decrease of the levels of transcripts for proteins
involved in photosynthesis. In tomato, the levels of the tran-
scripts for the nuclear-encoded small subunit of Rubisco
(RbcS) and chlorophyll binding protein (Cab) and Rubisco
activase (Rca) decrease in elevated [CO2], whereas the lev-
els of several plastid-encoded transcripts including RbcL,
PsaA, PsaB and PsbA (encoding the large subunit of
Rubisco and core proteins in photosystems I and II) do not
decrease (Van Oosten et al. 1994; Van Oosten & Besford
1995). In wheat, elevated [CO2] led to a decrease of the
transcripts for RbcS, RbcL and three other Calvin cycle
enzymes (Nie et al. 1995b). Recently, Cheng et al. (1998)
reported that long-term exposure to elevated [CO2] leads to
a decrease of the level of the RbcS and the RbcL transcripts
in Arabidopsis. Moore et al. (1998) reported that the level
of the RbcS transcript is decreased in elevated [CO2] in
parsley, bugle, cotton, sunflower, wheat, bean and radish. It
has been proposed that the acclimation of photosynthesis is
due to a sugar-mediated repression of Rubisco and other
proteins that are required for photosynthesis (Stitt 1991;
Jang & Sheen 1994; Van Oosten & Besford 1996).
Acclimation of photosynthesis to elevated [CO2] is
usually more marked in nitrogen-limited plants than in
well-fertilized plants (Wong 1979; Oberbauer et al. 1986;
Arp 1991; Radoglou, Alpho & Jarvis 1992; Tissue et al.
1993; Pettersson, MacDonald & Standenberg 1993; Sage
1994; Pettersson & MacDonald 1994; El-Kohen &
Mousseau 1994; Miglietta, Giuntole & Bindi 1996; Bowler
& Press 1996). Analysis of the gas exchange characteris-
tics indicates that the shift to a decreased carboxylation
capacity in elevated [CO2] is more marked in nutrient-
limited than in well-fertilized plants (Arp 1991; Sage
1994), and that elevated [CO2] leads to a larger decrease of
Rubisco in nitrogen-limited plants than in well-fertilized
plants (Wong 1979; Tissue et al. 1993; Delgrado et al.
1994; Webber, Nie & Long 1994; Riviere-Rolland,
Contard & Betsche 1996; Rogers et al. 1996; Ziska et al.
1996). Further, Riviere-Rolland et al. (1996) showed that
elevated [CO2] led to a decrease of Rubisco activity and
the RbcS transcript level in pea plants growing in limiting
nitrogen, but not when the plants were supplied with a high
supply of nitrogen, or were grown with limiting phosphate.

The latter treatment indicates that the decrease of Rubisco
in the nitrogen-limited plants was not just a non-specific
effect, due to slow rates of growth.
These results could be interpreted as evidence that sugar-
repression of Rubisco expression operates more effectively
in nitrogen-limited plants, which would explain the larger
decline of Rubisco and the accentuated acclimation of pho-
tosynthesis to elevated [CO2] in nitrogen-limited plants.
However, there is an alternative explanation: the decrease
of Rubisco may reflect a general decrease of leaf protein,
due to reallocation of nitrogen to younger leaves or earlier
leaf senescence in nitrogen-limited plants (Coleman,
McConnaughay & Bazzaz 1993; Nie et al. 1995a; Miller
et al. 1997). These processes may be accelerated in
elevated [CO2], because the plants are larger and therefore
experience a more acute nitrogen limitation (see Section
3.1.1. and Section 5 for further discussion).
2.2 Nitrogen-limited plants contain large amounts
of non-structural carbohydrates even in ambient
carbon dioxide and accumulate more in elevated
carbon dioxide
Elevated [CO2] typically leads to an increased content of
non-structural carbohydrates (Stitt 1991) but the extent of
the change varies, depending on the species and conditions
(Körner et al. 1995; Poorter et al. 1997; Tissue et al. 1997;
Körner et al. 1997). In general, the increase of starch or
other carbohydrate storage polymers is larger than the
increase of sugars (Morin, Andre & Betsche 1992; Den-
Hertog et al. 1996; Poorter et al. 1997; Baxter, Ashenden &
Farrar 1997; Fonseca, Bowsher & Stulen 1997; Tissue
et al. 1997; Moore, Palmquist & Seeman 1997), but the
extent of the changes of sucrose, reducing sugars and
starch vary considerably between species (see Bowler &
Press 1996; Tuba et al. 1996; Schappi & Körner 1997;
Moore et al. 1998).
Nitrogen limitation also leads to an increase of non-struc-
tural carbohydrates, which is mainly due to starch (Waring
et al. 1985; Hofstra, Lanting & De Visser 1985; Rufty,
Huber & Volk 1988; Fichtner & Schulze 1992; Fichtner
et al. 1993; Paul & Driscoll 1997; Scheible et al. 1997b).
Sugars remain relatively low and sometimes even decrease
in nitrogen-limited plants (Rufty et al. 1988; Fichtner et al.
1993; Reddy et al. 1996; Scheible et al. 1997c).
When nitrogen-limited plants are exposed to elevated
[CO2] there is a further accumulation of non-structural car-
bohydrates (Wong 1979, 1990; Bowler & Press 1996;
Baxter et al. 1997; Ferrario-Mery et al. 1997; Fischer et al.
1997; Geiger et al. 1999). The nitrogen supply may also
influence the relative extent of starch and sugar accumula-
tion in elevated [CO2]. Elevated [CO2] led to a large
increase of sugars whereas starch did not markedly
increase when Nicotiana plumbaginifolia was growing in
hydroponic culture in high nitrate, whereas it led to a large
increase of starch and a smaller increase of sugars when the
plants were growing in pots on a lower nitrogen supply
(Ferrario-Mery et al. 1997). A very marked increase of
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 585
starch in elevated [CO2] has also been reported in nitrogen-
limited sugar beet (Kutik et al. 1995) and Pinus palustris
(Pritchard et al. 1997). In tobacco, elevated [CO2] led to an
increase of starch and also of sucrose and reducing sugars
in nitrogen-sufficient plants, whereas it led to a further and
marked increase of starch but not of sucrose and reducing
sugars in nitrogen-limited plants (Geiger et al. 1999). On
the other hand, Bowler & Press (1996) found an increase of
sugars in elevated [CO2] when Agrostis capillaris and
Nardus stricta were nitrogen-limited, but not when they
received a high nitrogen supply. This may be because these
grasses accumulate fructans rather than starch.
2.3 The stimulation of plant growth is decreased
and eventually abolished as the nitrogen supply
is decreased
Whereas elevated [CO2] typically leads to a marked
increase of biomass in well-fertilized plants (Bowes 1996),
this response is modified when the nitrogen fertilization is
suboptimal. Biomass was not significantly increased by
elevated [CO2] in nitrogen-limited soybean (Sionit 1983),
tobacco (Geiger et al. 1998b), rice (Ziska et al. 1996),
Agrostis capillaris and Nardus strictus (Bowler & Press
1996), Calluna vulgaris, Erica tetralix, Molinia caerula,
Rumex obtusifolius, Arrhenatherum elatius (Arp 1998),
loblolly pine (Thomas, Lewis & Strain 1994; Johnson, Ball
& Walker 1995; Tissue et al. 1997), sweet chestnut (El-
Kohen & Mousseau 1994) and several other woody species
(Eamus & Jarvis 1989). Elevated [CO2] led to an increase
of biomass in nitrogen-limited wheat (Hocking & Meyer
1991a; Mitchell et al. 1993; McKee & Woodward 1994;
Rogers et al. 1996; Fangmeier et al. 1997), cotton (Wong
1979; Rogers et al. 1993), Xanthium occidentale (Hocking
& Meyer 1985), Vaccinium myrtillus (Arp 1991),
Eucalyptus grandis (Conroy, Milham & Barlow 1992), oak
seedlings (Norby et al. 1986), loblolly pine seedlings
(Gebauer, Reynolds & Strain 1996) and birch (Pettersson
et al. 1993) but the relative and absolute stimulation was
smaller than in well-fertilized plants. Occasionally,
elevated [CO2] produces a large stimulation of growth in
nitrogen-limited conditions, including Calluna vulgaris
(Whitehead, Caporn & Press 1997) and wheat (Wong &
Osmond 1991).
Although the variability may be partly due to species
differences, it is probably also a result of differences in the
degree of nitrogen deficiency and the way in which the
plants were fertilized (see Pettersson & MacDonald 1994).
When Eucalyptus spp. (Wong, Kriedermann & Farquhar
1992), birch (Pettersson & MacDonald 1994; Johnson
et al. 1995; Silvola & Ahlholm 1995), ponderosa pine
(Johnson et al. 1995), loblolly pine (Gebauer et al. 1996),
Agrostis capillaris, Nardus stricta (Bowler & Press 1996),
rice (Ziska et al. 1996), cotton (Rogers et al. 1993), wheat
(Hocking & Meyer 1991b; Rogers et al. 1996) and tobacco
(Geiger et al. 1999) were grown at three or more nitrogen
supplies, elevated [CO2] led to a large increase of biomass
at the highest nitrogen supply, a small increase of biomass
586 M. Stitt and A. Krapp
at moderately limiting nitrogen supply, whereas it had no
positive effect on biomass or even led to a slight decrease
of biomass at the lowest nitrogen supply. Different results
may be found in ecosystems (Lloyd & Farquhar 1996), but
this may reflect long-term interactions between the plants
and the soil which modifies the nitrogen supply that the
plant is receiving.
These results imply that the nitrogen use efficiency
(see Section 2.4) and/or nitrogen uptake and assimilation
(see Sections 2.5–2.7) can be increased in elevated
[CO2]. However, at a very low nitrogen supply, this is no
longer possible. Arp (1998) have proposed that when the
nitrogen supply is very low, the tissue nitrogen content is
at a minimum and cannot be reduced by further redistri-
bution of nitrogen. This appears plausible, although proof
will require a better understanding of what a ‘minimum’
nitrogen content is, and why it cannot be decreased
further.
2.4 Nitrogen use efficiency is increased in
elevated [CO2]
Innumerable studies have reported that the nitrogen use
efficiency (the rate of growth per unit of nitrogen in the
plant) increases in elevated [CO2] (see, e.g. Wong 1979;
Hocking & Meyer 1991a, b; Pettersson et al. 1993; Rogers
et al. 1993; McKee & Woodward 1994; Jacob et al. 1995).
This increase of the nitrogen use efficiency is, in part, due
to the lower nitrate content in elevated [CO2] (Purvis,
Peters & Hageman 1974; Yelle, Gosselin & Trudel 1987;
Hocking & Meyer 1985, 1991b). Problems in interpreting
the physiological significance of an increased nitrogen use
efficiency that are due to a decrease of the nitrate content
will be discussed in Section 5.1. However, the organic
nitrogen use efficiency (which excludes nitrate and ammo-
nium from the denominator) usually also increases in ele-
vated [CO2] (Sage, Sharkey & Seeman 1990; Pettersson
et al. 1993; Baxter et al. 1994).
In elevated [CO2], a given rate of photosynthesis can be
achieved with lower activities of Rubisco, other Calvin
cycle enzymes, and light reaction components because
photorespiration is decreased (Stitt 1991; Quick et al.
1993; Woodrow 1994). This, in principle, could permit
decreased investment of nitrogen in the nitrogen-intensive
processes of photosynthesis and photorespiration in leaves
(see Section 2.1), releasing nitrogen for investment in other
processes and tissues. However, it is not yet clear if the
increased organic nitrogen use efficiency is solely due to
Rubisco, which is a major part of the total leaf protein,
operating more efficiently thanks to the higher [CO2] con-
centration, or if there has been an acclimation resulting in
re-allocation of nitrogen to other proteins in the plant.
In transgenic plants expressing an antisense RbcS
construct, decreased expression of Rubisco indeed leads to
reallocation of organic nitrogen to other proteins in the leaf,
including thylakoid proteins resulting in a higher organic
nitrogen use efficiency (Makino et al. 1997) and increased
rates of photosynthesis in low light (Lauerer et al. 1993; Stitt
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
& Schulze 1994). In elevated [CO2], although numerous
studies have reported a decrease of Rubisco (see Section
2.1), few studies have extended the analyses to determine
whether there is a selective increase of other proteins. The
decrease of Rubisco in elevated [CO2] is accompanied by
an increase in the amounts of other leaf proteins in pea
(Riviere-Rolland et al. 1996) and wheat (Rogers et al.
1996). The nitrogen released from Rubisco and other pro-
teins of the photosynthesis apparatus could alternatively be
allocated to promote the growth of non-photosynthetic
organs. However, even though elevated [CO2] stimulates
root growth it also leads to an increase of the whole plant
leaf area (Wong 1979), and there is no consistent evidence
for a selective increase of root growth relative to shoot
growth in elevated [CO2], or a selective change in the whole
plant leaf area (Pettersson et al. 1993; King, Thomas &
Strain 1996; Gebauer et al. 1996; Tissue et al. 1997; Baxter
et al. 1997; Drake et al. 1997).
2.5 Elevated carbon dioxide leads to a decrease
of the nitrogen content in nitrogen-limited plants,
but not necessarily in well-fertilized plants
The overall nitrogen concentration usually decreases
when plants are grown in elevated [CO2] (Wong 1979;
Curtis, Drake & Whigham 1989; Garbutt, Williams &
Bazzaz 1990; Coleman et al. 1991; Coleman & Bazzaz
1992; Hocking & Meyer 1991a, 1991b; Gries, Kimball &
Idso 1993; Pettersson et al. 1993; Pettersson &
MacDonald 1994; Luo, Field & Mooney 1994; Körner &
Miglietta 1994; Newberry et al. 1995; Poorter et al. 1997;
Ferrario-Mery et al. 1997; Tissue et al. 1997; Cotrufo,
Ineson & Scott 1998). The decrease of the nitrogen con-
tent and carbon/nitrogen ratio in elevated [CO2] is partly
an arithmetic consequence of the increase of non-struc-
tural carbohydrates. Plants in elevated [CO2] accumulate
more starch (see above) which can account for up to
40–50% of the leaf weight, and will automatically lead to
an apparent decrease in the concentrations of other leaf
components. However, the nitrogen concentration usually
decreases in elevated [CO2], even after correcting for this
effect (see Tissue et al. 1997; Poorter et al. 1997 and other
references above).
There have been very few systematic investigations of
the changes in the individual nitrogen-containing com-
pounds. Elevated [CO2] often leads to a marked decrease of
nitrate (Purvis et al. 1974; Yelle et al. 1987; Hocking &
Meyer 1985, 1991b; Ferrario-Mery et al. 1997; Geiger et al.
1998b). There have been hardly any studies of the changes
in ammonium and amino acid levels. Elevated [CO2] leads
to a decrease of ammonium and of the overall amino acid
levels in N. plumbaginifolia and tobacco (Ferrario-Mery
et al. 1997; Geiger et al. 1999). Protein typically decreases
by 10–20% in elevated [CO2] (e.g. Jacob et al. 1995; Nie
et al. 1995a; Sicher, Kremer & Bunce 1995; Ferrario-Mery
et al. 1997; Geiger et al. 1998b). In a study of 27 species,
Poorter et al. (1997) found that the decrease of leaf protein
in elevated [CO2] (on average 18%) was slightly smaller

than the decrease of total nitrogen (on average, 21%) and
of nitrate (on average, 22%).
The overall decrease of the nitrogen concentration in
elevated [CO2] has been interpreted in various ways: it
might reflect a higher nitrogen use efficiency due to reallo-
cation of protein (see Section 2.4), or ontogenetic drift
leading to accelerated senescence as a result of the faster
growth in elevated [CO2], inadequate nitrogen fertiliza-
tion, or inadequate rates of nitrogen uptake and assimila-
tion in elevated [CO2] (see Section 2.6).
With respect to the latter suggestion, several studies
have reported that elevated [CO2] has little or no effect on
protein content in young leaves but leads to an earlier
decrease in older leaves (Besford, Ludwig & Withers
1990; Nie et al. 1995a; Van Oosten et al. 1994; Miller et al.
1997). Coleman et al. (1993) showed for Abutilon
threophrasti that elevated [CO2] leads to a decrease of the
nitrogen concentration when plants of the same age are
compared, but not when plants of the same size are com-
pared, and argued that the carbon-dioxide-induced reduc-
tion in plant nitrogen concentration is not due to changes in
the plant nitrogen use efficiency but is more likely to be a
size-dependent phenomenon resulting from accelerated
plant growth. On the other hand, Roumet et al. (1996) still
found a decrease in the nitrogen concentration in elevated
[CO2] in 11 different species when they were compared at
a common dry weight after growth in ambient and elevated
[CO2]. Interpretation of age-dependent changes in the
nitrogen concentration requires detailed information about
the nutrient supply and the nutrient status of the plant, in
order to distinguish between true ontogenetic changes that
are linked to internal developmental programmes, and
plant-size effects that are related to the exhaustion of the
nutrient supply.
Significantly, the nitrogen supply has a marked impact
on the effect of elevated [CO2] on the nitrogen concentra-
tion in the plant. When wheat and cotton (Rogers et al.
1993, 1996), Avena fatua and Plantago erecta (Chu, Field
& Mooney 1996), Nardus stricta and Agrostis capillaris
(Bowler & Press 1996), Poa alpina (Baxter et al. 1997)
and tobacco (Geiger et al. 1999) were grown in elevated
[CO2], there was a large decrease of the leaf nitrogen con-
centration when the plants were grown at a low nitrogen
supply, whereas there was only a small and even non-sig-
nificant decrease when the plants were grown at a high
nitrogen supply. Arp (1991) and Poorter et al. (1997) noted
that the decrease in total leaf nitrogen in elevated [CO2]
was smaller when plants were grown in hydroponics than
in pots, which might again be related to the improved nitro-
gen nutrition in hydroponic culture. Elevated [CO2] actu-
ally led to a small statistically non-significant increase of
the nitrogen/carbon ratio and the nitrate and amino acid
content when N. plumbaginifolia was grown on high
nitrate in hydroponic culture (Ferrario-Mery et al. 1997).
Elevated [CO2] also led to a marked and statistically sig-
nificant increase of the nitrogen concentration and the
nitrate, amino acid and protein content of young nitrogen-
sufficient tobacco seedlings growing in sand-culture on a
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 587
high and frequently renewed nitrate supply, but not in older
plants (Geiger et al. 1998b). Further, elevated [CO2] stimu-
lated the current growth rate in the young tobacco
seedlings, but not in the older plants (Geiger et al. 1998b).
These results strongly indicate that the widely reported
decrease of the nitrogen concentration in elevated [CO2]
may be linked to the accessibility of nitrogen rather than a
direct or necessary consequence of the elevated [CO2] (see
Section 5.1 for further discussion).
2.6 Elevated carbon dioxide has no consistent
effect on nitrate and ammonium uptake
The decreased nitrogen concentration frequently observed
in elevated [CO2] (see Section 2.5) indicates that nitrate
uptake and assimilation often fail to keep pace with photo-
synthesis and growth in elevated [CO2]. The effect of ele-
vated [CO2] on whole plant net nitrogen uptake is
revealed more directly by comparing the change of the
nitrogen concentration with the change of plant biomass.
In some studies, the nitrogen content per plant still
increased in elevated [CO2] even though tissue nitrogen
concentration or the nitrogen/carbon ratio decreased
(Wong 1979; Hocking & Meyer 1991b; Chu, Coleman &
Mooney 1992; Tissue et al. 1997). In other studies, espe-
cially when the nitrogen supply was very low, the total
amount of nitrogen per plant was unaltered (Norby et al.
1986; O’Niell, Lumore & Norby 1987; Garbutt et al.
1990; Hocking & Meyer 1991a; Van Ginkel, Gorissen &
Van Veen 1997) or reduced (Coleman & Bazzaz 1992;
Conroy et al. 1992) in elevated [CO2].
Taken together, these results indicate that elevated [CO2]
can lead to increased nitrogen uptake, provided there is an
adequate supply of nitrogen outside the plant. As discussed
in the following paragraphs, elevated [CO2] could modify
inorganic nitrogen uptake by altering the access to or the
accessibility of nutrients in the soil, and/or by altering the
rates of uptake per unit of root surface.
Plants growing in elevated [CO2] possess larger root sys-
tems, at least in absolute terms (Pettersson et al. 1993;
Jacob et al. 1995; Jackson & Reynolds 1996; see also
Drake et al. 1997), which should allow them to exploit a
larger soil volume and might be expected to promote
nitrate and ammonium uptake. In addition to an overall
increase in root biomass (see above), elevated [CO2] has
been reported to increase lateral and secondary root forma-
tion in birch (Pettersson et al. 1993), loblolly pine
(Larigauderie, Reynolds & Strain 1994), and ponderosa
pine (Tingey et al. 1996). Elevated [CO2] altered the distri-
bution of root growth in wheat, promoting root growth in
the upper soil layers (Van Vuuren et al. 1997). However,
the beneficial effect of an increased root volume might be
counteracted by the effects of elevated [CO2] on water
flow. Increased [CO2] usually leads to partial closure of
stomata, resulting in lower transpirative water flow. This
may be advantageous under water-limiting conditions
because it leads to a higher water use efficiency, but it will
decrease the mass flow of water in the soil to the roots and
588 M. Stitt and A. Krapp
might therefore decrease the root surface concentrations of
soil-mobile minerals, including nitrate (Conroy & Hocking
1993; Van Vuuren et al. 1997). This is unlikely to lead to
nitrate becoming limiting in well-fertilized soils, but could
decrease access to nitrate when the soil nitrate solution is
very dilute. Increased exudation of carbon and trapping of
nitrogen in the soil microflora might also modify soil nitro-
gen availability, especially when overall concentration is
low (Schenk et al. 1995).
The reported effect of elevated [CO2] on the rate of
nitrate or ammonium uptake per unit root weight is also
rather variable. Elevated [CO2] increased the rate of
nitrate uptake per unit root weight in loblolly pines
(Bassirirad et al. 1996) and Prosopis glandulosa
(Bassirirad et al. 1997), did not alter the rate of nitrate
uptake in Nardus agrostis (Bassirirad et al. 1997), and
decreased nitrate uptake in a mixed field community
(Jackson & Reynolds 1996). Ammonium uptake was not
altered in elevated [CO2] in loblolly pine (Bassirirad et al.
1996), whereas it was increased in a mixed field commu-
nity (Jackson & Reynolds 1996). The response of nitrate
and ammonium uptake to elevated [CO2] may depend on
the nitrogen concentration supplied. Whereas elevated
[CO2] led to increased rates of nitrate uptake in loblolly
pine growing at high nitrate, it led to decreased uptake in
plants growing on low nitrate (Larigauderie et al. 1994).
Interpretation of these results on the effect of elevated
[CO2] on nitrate and ammonium uptake is complicated by
the dynamics of nitrate in the soil, and by the multiple
transport systems for nitrate, and the complex regulation
of nitrate uptake (see Section 3.1).
2.7 There is no consistent evidence for a
stimulation of nitrate and ammonium
assimilation in elevated carbon dioxide
Nitrate is reduced in the cytosol via nitrate reductase (NR)
to nitrite, which enters the plastid and is reduced to ammo-
nium via nitrite reductase. Glutamine synthetase (GS) then
incorporates ammonium into glutamate to form glutamine,
and ferredoxin-dependent glutamine synthase (Fd-
GOGAT) in a reductive transamination transfers the amino
group from glutamine to 2-oxoglutarate to form two
molecules of glutamate. Aminotransferases then transfer
the amino group from glutamate to aspartate and alanine.
Glutamate, aspartate and occasionally glutamine act as the
amino donors for the synthesis of other amino acids,
nucleotides and other nitrogen-containing compounds in
the cell. The major regulatory site in nitrate assimilation is
thought to be at NR (see Section 3.2). Nitrate assimilation
occurs mainly in the leaves in herbaceous plants, but a
larger proportion is assimilated in the roots in many woody
species (Andrews 1986; Andrews et al. 1992). De novo
ammonium assimilation occurs mainly in the roots
(Marschner 1995).
Until recently, there was no consistent evidence for an
increase of NR activity in elevated [CO2]. Although elevated
[CO2] led to a small increase of NR activity in mustard
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
(Maevskaya et al. 1990) and Vigna radiata (Sharma &
Sen Gupta 1990), it produced a two-fold decrease of NR
activity in wheat (Hocking & Meyer 1991a, 1991b) and
maize (Purvis et al. 1974), a 25% decrease in N.
plumbaginifolia grown in vermiculite (Ferrario-Mery
et al. 1997), and a 15% decrease in N. plumbaginifolia
grown in hydroponic culture (Ferrario-Mery et al. 1997).
Elevated [CO2] also led to a 30% decrease of nitrite reduc-
tase activity in lettuce (Besford & Hand 1989), which is
expressed co-ordinately with NR (Hoff, Truong &
Caboche 1994; Vincentz et al. 1993).
It is difficult to understand these results, for two rea-
sons. Firstly, a higher rate of nitrate assimilation will pre-
sumably be required in elevated [CO2] to support the
higher rate of plant growth. As already discussed, even
though nitrogen decreases on a weight basis in elevated
[CO2] this is partly due to a decrease of nitrate (Purvis
et al. 1974; Yelle et al. 1987; Hocking & Meyer 1985,
1991b), and the organic nitrogen content per plant often
increases (see, e.g. Wong 1979; Hocking & Meyer 1991a,
1991b). Secondly, based on our current knowledge about
the regulation of nitrogen metabolism (see Section 4.2),
we would expect that elevated [CO2] should stimulate
rather than inhibit nitrate assimilation.
Recently, Fonseca et al. (1997) showed that transfer of
Plantago major to elevated [CO2] leads to a short-term
increase of the Nia transcript level in leaves and, espe-
cially, in the roots during the first 4 d in elevated [CO2],
that is accompanied by a transient 50% increase of NR
activity in the shoot and a doubling of NR activity of the
roots. Geiger et al. (1998b) showed that long-term expo-
sure of tobacco to elevated [CO2] leads to a sustained
increase of NR activity due to subtle changes in the diurnal
regulation of NR activity in the leaves and the roots (see
Section 5.2.2 for further discussion).
Few studies of elevated [CO2] have differentiated
between the use of nitrate and ammonium as nitrogen
sources. In principle, elevated [CO2] might favour ammo-
nium assimilation in leaves because photorespiration and
the associated recycling of ammonium is decreased (see
Sections 3.2.2 and 5.2.3). The levels of the transcripts for
the plastid GS and for Fd-GOGAT did not decrease in
high [CO2] in tobacco leaves (Migge et al. 1997), plastid
GS protein was only slightly decreased, and Fd-GOGAT
protein was unaffected. Elevated [CO2] also had no effect
on overall GS activity in the leaves of N. plumbaginifolia
(Ferrario-Mery et al. 1997). The GOGAT pathway
capacity that is released by the decreased rate of pho-
torespiration is therefore available for net ammonium
assimilation, or for assimilation of ammonium formed
during nitrate assimilation in the leaves. Elevated [CO2]
might additionally promote ammonium assimilation in
the roots, because more carbohydrate is available to sup-
ply energy and carbon skeletons. Elevated [CO2] leads to
a three- to four-fold increase of overall GS activity in the
roots of tobacco growing on limiting ammonium nitrate
in elevated [CO2], especially in nitrogen-limited plants
(Geiger et al. 1999).

Summarizing, there is a strong interaction between the
nitrogen supply and the response of photosynthesis,
metabolism and growth to elevated [CO2]. On the one
hand, the acclimation of photosynthesis is accentuated and
the stimulation of growth in elevated [CO2] is strongly cur-
tailed in nitrogen-limited plants. On the other hand, ele-
vated [CO2] often leads to a decrease of the internal
nitrogen levels. The changes of nitrogen metabolism in ele-
vated [CO2] have, however, not been studied in detail and
they have not been related to our knowledge of the regula-
tion of nitrogen metabolism and its interaction with carbon
metabolism and growth processes. This makes it difficult
to distinguish between possible explanations in which
changes in the nitrogen status are seen simply as a change
in a resource that is needed for growth, or as a factor that
modulates sugar-mediated changes in gene expression,
metabolism and growth, or in a more general way, modi-
fies allocation, ontogeny or senescence. The following two
sections discuss the emerging evidence from experiments
in model systems that nitrate and other nitrogen-containing
compounds act as an important source of signals to regu-
late plant metabolism and growth, and consider how car-
bon metabolism acts to modulate nitrogen uptake,
assimilation and utilization.
3 MANY ASPECTS OF PLANT METABOLISM
AND GROWTH ARE REGULATED BY SIGNALS
THAT DERIVE FROM NITRATE OR NITROGEN
METABOLISM
Application of nitrate fertilizer leads to increased rates of
nitrate uptake (Jackson, Flesher & Hageman 1973;
Clarkson & Lüttge 1991), increased levels of nitrate in the
plant, increased activities of NR (Shaner & Boyer 1976;
Galangau et al. 1988), nitrite reductase (Wray 1993), GS
and Fd-GOGAT (Sakakibara et al. 1991), increased rates
of nitrate assimilation resulting in higher levels of ammo-
nium, amino acids, protein and other nitrogen-containing
constituents in the plant (Marschner 1995; Scheible et al.
1997a, 1997b) increased levels of organic acids (Martinoia
& Rentsch 1994) and decreased levels of starch (Hofstra
et al. 1985; Fichtner & Schulze 1992; Scheible et al.
1997a). The rate of growth is increased, and there are
marked changes in allocation resulting in preferential
growth of the shoot compared with the roots (Brouwer
1962; Bloom, Chapin & Mooney 1985; Lambers et al.
1990), thicker and less branched roots (Grime et al. 1991;
Fichtner & Schulze 1992; Marschner 1995) and delayed
flowering (Bernier et al. 1993). Localized application of
nitrate to part of the root system stimulates lateral root
growth at that site (Drew & Saker 1975; Granato & Raper
1989; Lainé, Ourry & Boucard 1995; Sattelmacher &
Thoms 1995). Nitrate also stimulates seed germination
(Hilhorst & Karssen 1989).
It has long been suspected that nitrate is not only a
resource but also acts, directly or indirectly, to trigger sig-
nals that modulate gene expression, metabolism and devel-
opment (Redinbaugh & Campbell 1991; Crawford 1995;
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 589
Hoff et al. 1994; Daniel-Vedele & Caboche 1996; Daniel-
Vedele, Filleur & Caboche 1998; Stitt & Scheible, 1998).
By analogy with micro-organisms, we can expect that
nitrogen metabolites such as ammonium, glutamine and
asparagine will also act in an analogous manner. If this is
the case, the changes in the levels of these compounds in
elevated [CO2] (see Section 2.5) can be expected to have
profound effects on metabolism and growth. The following
section will discuss recent advances in understanding the
processes involved in nitrate and ammonium uptake and
assimilation, and the significance and possible mechanisms
of nitrogen-signalling processes in plants. Genotypes with
low activity of NR have provided an invaluable experimen-
tal tool to dissect nitrogen signalling, because they allow
the supply and the internal level of nitrate to be varied inde-
pendently of the rate of nitrate assimilation, the levels of
amino acids and protein in the plant, and the overall rate of
plant growth (Scheible et al. 1997a, 1997b).
3.1. Nitrate uptake
3.1.1 Physiological studies
Based on studies of the multiphasic saturation kinetics, it has
been proposed that net nitrate uptake can be mediated by
several different systems including a high affinity nitrate-
inducible transporter and various low affinity nitrate-
inducible transporters (Aslam, Travis & Huffacker 1992;
King et al. 1993; Aslam, Travis & Rains 1996), and reversed
by a nitrate efflux system (Aslam et al. 1996; Volk 1997).
Nitrate uptake is strongly stimulated after pre-incuba-
tion with nitrate. Studies of the influence of pre-incuba-
tion on nitrate uptake kinetics indicate that the high
affinity transporter and one low affinity nitrate-inducible
transport system are stimulated by nitrate, and that
another low affinity transporter is constitutive (Aslam
et al. 1992; King et al. 1993; Aslam et al. 1996). Pre-
incubation with nitrate also increases the rate of nitrate
efflux (Aslam et al. 1996; Volk 1997).
Nitrate uptake is subject to feedback regulation, and is
therefore inhibited by prolonged incubation with nitrate, or
by addition of ammonium. It has been suggested that this
decrease may be due to feedback effects that are mediated
by nitrate itself, or by ammonium or certain amino acids
(see Siddiqi et al. 1990; Clarkson & Lüttge 1991; Muller &
Touraine 1993; King et al. 1993; Rufty et al. 1993;
Imsande & Touraine 1994). Evidence for a feedback regu-
lation of nitrate uptake by down-stream products formed
during nitrate assimilation has recently been provided by
Gojon et al. (1998), who showed that tobacco and N.
plumbaginifolia transformants with increased expression
of NR and consequently increased levels of glutamine have
a lower nitrate uptake rate than wild-type plants.
On the other hand, large amounts of nitrate accumulate
in NR-deficient mutants and transformants of barley
(Warner & Huffaker 1989, N. plumbaginifolia (Ferrario
et al. 1995), tobacco (Scheible et al. 1997a) and
Arabidopsis (Meyer zu Hoerste 1998), indicating that
590 M. Stitt and A. Krapp
accumulation of nitrate does not strongly inhibit its own
uptake, at least in these species. Lauerer (1996) found up to
50% higher rates of nitrate uptake per unit root weight in
tobacco transformants with very low NR activity. Gojon
et al. (1998) found unaltered or only slightly lower nitrate
uptake rates in N. plumbaginifolia and tobacco genotypes
with a small decrease in NR activity, again indicating that
high internal nitrate does not lead to a strong feedback inhi-
bition of nitrate uptake, although, as discussed by Gojon
et al. (1998), the lower levels of nitrate assimilation in
these genotypes would be expected to lead to a stimulation
of nitrate uptake and this could be masking a weak feed-
back regulation of nitrate uptake by nitrate.
There is considerable species-dependent variation with
respect to the amount of nitrate that accumulates inside the
plant. Large amounts accumulate in tobacco and
Arabidopsis (Scheible et al. 1997a; Schulze et al. 1994),
smaller amounts in pea (Lexa & Cheeseman 1997) and
Plantago major (Fonseca et al. 1997), and very low
amounts in rice (Makino et al. 1997). This indicates that
the feedback mechanisms regulating nitrate uptake may
vary, depending on the species.
3.1.2 Molecular studies
These physiological studies of nitrate uptake have recently
been extended by the molecular cloning of the transporters
involved in nitrate uptake. These advances mean that the
regulation of nitrate uptake can now be subjected to a
molecular analysis (see Daniel-Vedele et al. 1998, and
Forde & Clarkson 1999 for more details).
A gene encoding a low-affinity, passive nitrate uptake
system (Chl-1, now renamed Nrt1 : 1At) was cloned from a
T-tagged chlorate-insensitive Arabidopsis mutant (Tsay
et al. 1993). Chl-1 is preferentially expressed in roots, and
is induced by nitrate. The precise role of this transporter is
unclear, however, because the Chl-1 mutant shows
pleiotropic changes in cellular pH and in the levels of other
inorganic ions. Since the Chl-1 mutant shows decreased
rates of nitrate uptake in the presence of ammonium, but
not when nitrate is given as the sole nitrogen source
(Huang et al. 1996), it has been proposed that there may be
further ammonium-repressed low affinity transporters that
are able to substitute for the CHL-1 protein in the absence
of ammonium. Recently, Lauter et al. (1996) cloned two
CHL-1 homologs in tomato, and showed that one of them
was expressed constitutively in root cells and root hairs,
whereas the other was induced by nitrate and expressed
specifically in root hairs.
More recently, sequence homology to the Aspergillus
nitrate transporter and Chlamydomonas high-affinity
nitrate transporters (Quesada, Galvan & Fernandez 1994)
has allowed cloning of genes that encode the high-affinity
nitrate transporters in barley (Trueman, Richardson &
Forde 1996) and N. plumbaginifolia (Quesada et al. 1997).
Genomic analyses indicate the presence of a small multi-
gene family in barley (Trueman et al. 1996). The expres-
sion of the high affinity Nrt2 : 1 transporter has been
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
investigated in N. plumbaginifolia and tobacco (Krapp
et al. 1998). Expression is restricted to the roots. Nitrate
addition leads to a rapid but transient increase of the tran-
script level in wild-type roots. The contribution of feed-
back from nitrate itself and from products of nitrate
assimilation was dissected using tobacco transformants
with very low NR activity. In these plants, nitrate addition
led to a strong and sustained increase of the level of the
Nrt2 : 1 transcript, whereas ammonium addition led to a
strong decrease. This implies that Nrt2 : 1 is repressed by
ammonium or compounds produced from ammonium,
but not by nitrate accumulation. There were also marked
diurnal changes of the Nrt2 : 1 transcript level in the roots
of wild-type tobacco plants, with a maximum in the first
part of the light period and a decrease later in the pho-
toperiod (A. Krapp. W.-R. Scheible, M. Caboche, unpub-
lished results). These diurnal changes of the Nrt2 : 1
transcript level were modified in mutants with lower
expression of NR, and were positively correlated with the
diurnal changes in NR protein and activity and negatively
correlated with ammonium, glutamine and malate
accumulation in the leaves. It will be important to
identify the precise signals that link Nrt2 : 1 expression to
nitrogen metabolism in the shoot, to investigate how
rapidly these changes in transcript translate into changes
in transporter protein, and also to investigate whether
there are faster changes in root uptake or xylem loading
due to post-translational regulation or regulation by low
molecular weight effectors.
These physiological and molecular studies demonstrate
that there is a close interaction between nitrate uptake, and
the metabolism of nitrate to further compounds in the
plant. The finding that nitrate uptake and Nrt2 : 1 expres-
sion are insensitive to nitrate accumulation but very sensi-
tive to feedback by down-stream signals generated as a
result of nitrate assimilation explains why nitrate can be
accumulated to relatively large levels in tobacco in excess
of that required for current growth. This will, of course,
also depend on an effective regulation of nitrate assimila-
tion, to prevent accumulation of down-stream metabolites
which inhibit nitrate uptake.
3.2 Nitrate assimilation
Shaner & Boyer (1976) observed that NR activity
increases dramatically when plants are re-fertilized with
nitrate. Subsequent research has shown that nitrate assimi-
lation is subject to transcriptional, post-transcriptional and
post-translational regulation at the step catalysed by NR
(Hoff et al. 1994; Crawford 1995; Nussaume et al. 1995;
Daniel-Vedele et al. 1998).
3.2.1 Regulation of Nia transcription
The Nia transcript and NR protein are very low in
nitrate-limited plants or in the presence of ammonium,
and high in plants grown on high nitrate (Hoff et al.
1994; Scheible et al. 1997c). These transcripts also show

a marked diurnal rhythm. In nitrate-replete tobacco and
tomato, the Nia transcript is present at a high level at the
end of the night, stays high or rises slightly during the
first 1–2 h in the light, declines markedly during the
remainder of the photoperiod, and recovers gradually
during the dark period (Galangau et al. 1988; Scheible
et al. 1997c; Geiger et al. 1998b). NR protein and activity
increase by two- to three-fold in the first 4 h of the light
period, decrease during the remainder of the light period
and the first part of the dark period, and start to recover
during the last part of the night (Galangau et al. 1988;
Scheible et al. 1997c; Geiger et al. 1998b). The changes
of NR protein occur with a slight time delay compared
with the changes of the Nia transcript level, as expected if
the changes in the Nia transcript level contribute to the
diurnal changes in NR protein.
Nia transcription is induced by nitrate (Pouteau et al.
1989; Cheng et al. 1992; Lin et al. 1994) and repressed by
glutamine (Hoff et al. 1994). These nitrogen metabolites
probably play a major role in the regulation of Nia
expression. The decrease of the Nia transcript during the
photoperiod is correlated with a decrease of nitrate
(Scheible et al. 1997c) and an accumulation of glutamine
(Scheible et al. 1997c), and the gradual recovery during
the night is correlated with a decrease of glutamine and a
gradual increase of nitrate. Sugar-mediated regulation
could also contribute to the light-induced changes of the
Nia transcript (see Section 4.2). The transcript for nitrite
reductase is regulated in a similar manner, except that
nitrite reductase activity is about 10-fold higher than NR
activity (Hoff et al. 1994). This co-ordinate regulation
presumably ensures that nitrite, which is toxic, is con-
verted through to ammonium.
3.2.2 Post-translational regulation of NR
Faster changes due to reversible post-translational regula-
tion of NR are superimposed on these changes of NR pro-
tein. When leaves are darkened, NR is inactivated via a
two-step process that involves phosphorylation of ser543
and the subsequent magnesium-dependent binding of an
inhibitory 14–3-3 protein to NR (Bachmann et al. 1996;
Moorhead et al. 1996).
There is substantial evidence that NR activation is regu-
lated in response to sugar-mediated signals (see Section
4.2.2). Until recently, however, there was no evidence that
signals from nitrogen metabolism affect the post-transla-
tional regulation of NR. Scheible et al. (1997c) showed
that dark inactivation of NR is partially or completely
reversed in nitrate-limited wild-type plants and in mutants
and transformants with decreased expression of NR. This
abolition of dark inactivation was correlated with a
decreased level of glutamine and ammonium which are the
products of nitrate assimilation, and NR activation could
be decreased by feeding glutamine to detached leaves
(Scheible et al. 1997c; Morcuende et al. 1998). Nitrate
does not affect the activation state of NR (Ferrario,
Valadier & Foyer 1996).
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 591
3.2.3 Post-transcriptional regulation
There is evidence for further, uncharacterized, mechanisms
that modulate either the translation of the Nia transcript
and/or rate of the degradation of NR protein. For example,
NR protein still decreases in the second half of the pho-
toperiod in transformants that express Nia constitutively
under the control of the 35S promotor (Vincentz &
Caboche 1991; Ferrario et al. 1995). The decline of NR
protein is absent in the second part of the photoperiod in
mutants that have a lower number of functional Nia genes,
even though the Nia transcript level declines in the same
way as in wild-type plants (Scheible et al. 1997c). Similar
changes are seen in elevated [CO2] (see Section 5.3). There
is also evidence that nitrite reductase is subject to post-
transcriptional regulation (Crete, Caboche & Meyer 1997).
Protein phosphorylation also may act as a trigger for
protein degradation. When a modified form of NR with a
truncated N-terminus that was not susceptible to post-
translational dark inactivation was overexpressed, the
resulting protein did not decline in the second part of the
photoperiod (Nussaume et al. 1995). There is also a cor-
relation between the phosphorylation state or the activa-
tion state of NR and the rate at which NR protein
decreases (Kaiser & Huber 1997; Scheible et al. 1997c;
Geiger et al. 1998b).
3.3 Ammonium uptake and assimilation
3.3.1 Ammonium uptake
Ammonium uptake exhibits multiphasic saturation kinetics
with low and high affinity components (Ullrich et al. 1984;
Wang et al. 1993). A high affinity ammonium transporter
has been cloned in Arabidopsis (Ninneman, Jauniaux &
Frommer 1994) and tomato (Lauter et al. 1996).
Expression was organ-specific, and transcript for the
tomato ammonium transporter LeAmt1 was found in roots,
but not in leaves or stems. Within the roots, the LeAmt1
transcript was found in root hairs but also in other root tis-
sues. The transcript level was not markedly decreased by
nitrate or increased by ammonium (Lauter et al. 1996).
3.3.2. Ammonium assimilation
Ammonium can be assimilated via the plastid GS2 or a series
of cytosolic GS1 isoforms (Lam et al. 1996). Cytosolic GS is
present as isoforms that show a cell specific distribution
(Lam et al. 1996; Dubois et al. 1996). One isoform of the
cytosolic GS is phloem specific (Lam et al. 1996).
Any consideration of ammonium assimilation in leaves
is complicated by the interaction between de novo nitrate
and ammonium assimilation, and the recycling of the
ammonium which is released in leaves during photorespi-
ration. Up to 90% of the ammonium assimilated in the
leaves of a C3 plant in ambient [CO2] represents to refixa-
tion of photorespired ammonium, rather than de novo
assimilation of nitrate or ammonium. Arabidopsis mutants
with null activity of the plastid GS were able to grow when
592 M. Stitt and A. Krapp
photorespiration was suppressed in 2–5% carbon dioxide,
but died in ambient [CO2] (Somerville 1986; Lea & Forde
1994; Lam et al. 1996). The cytosolic GS activities are
obviously too low, or have an inappropriate location, to
cope with the fluxes of ammonium during photorespira-
tion. Incidentally, this does not mean that the plastid GS
does not contribute to de novo ammonium assimilation in
wild-type plants, indeed it is difficult to see how the
ammonium formed by nitrite reductase in the plastid can
be separated from the plastid GS.
There is substantial evidence that nitrogen signalling
modulates the expression of the GOGAT pathway. Nitrate
leads to an increased level of the transcripts for Gln1 (encod-
ing the cytosolic glutamine synthetase, GS1), Gln2 (encod-
ing plastid glutamine synthetase; GS2) and Glu
(ferredoxin-dependent glutamate synthase; Fd-GOGAT) in
maize roots (Redinbaugh & Campbell 1993), tobacco roots
and tobacco leaves (Scheible et al. 1997b), and of the Glu
transcript in illuminated barley leaves (Pajuelo et al. 1997).
The changes of the transcript for the cytosolic GS were
much more marked than for plastid GS, and included the
appearance of a second Gln1 transcript (Scheible et al.
1997b). The increase of the GS transcript levels after adding
nitrate was accompanied by an increase of overall GS activ-
ity in the shoots and roots (Scheible et al. 1997b). Migge
et al. (1997) reported that nitrate addition resulted in the
appearance of a second form of the plastidic GS in tomato
leaves, which was absent when tomato plants were grown on
ammonium as the sole nitrogen source. In maize roots,
nitrate led to an increase of one GS isoform, and of Fd-
GOGAT (Sivasankar & Oaks 1996). In anaerobic rice
seedlings, nitrate led to an increase of Fd-GOGAT protein,
but not of the cytosolic or plastidic GS (Mattana et al. 1996).
Several lines of evidence show that the GOGAT path-
way is probably induced in response to nitrate, rather than
the assimilation of nitrate. The transcripts for Gln2, Glu
and to a lesser extent, Gln1 also increase when nitrate is
supplied to NR-deficient genotypes or genotypes with very
low expression of NR (Vaucheret et al. 1990;
Kronenberger et al. 1993; Scheible et al. 1997b), and this
was accompanied by a marked increase in overall GS
activity in the roots though not in the shoots. Addition of
tungsten, which blocks the synthesis of the molybdenum-
pterin cofactor and inhibits NR activity, had no marked
effect on the levels of transcript and protein for plastid GS
and Fd-GOGAT (Migge et al. 1997). Further, glutamine
and asparagine also had no effect on GS and Fd-GOGAT
activity in maize roots even though they led to a marked
decrease of NR activity (Sivasankar & Oaks 1996).
3.4 Provision of reducing equivalents for nitrate
assimilation
When nitrate is assimilated in leaves in the light, the reduc-
ing equivalents are delivered by photosynthetic electron
transport. There is ample evidence that addition of nitrate
leads to an increase rate of linear electron transport (Foyer
et al. 1994a, 1994b). In the alga Selenastrum minutum,
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
this leads to a severe oxidation of the photosynthetic elec-
tron chain, and reverses the thioredoxin-mediated activa-
tion of the Calvin cycle enzymes resulting in a marked
inhibition of photosynthesis (Huppe & Turpin 1994;
Turpin, Weger & Huppe 1997). In addition to this direct
effect, nitrate may also exert further regulatory action on
electron transport. Lauerer (1996) observed that accumula-
tion of nitrate in transformants with low NR activity was
accompanied by an increase of the chlorophyll b/chloro-
phyll a ratio, indicating that nitrate-mediated signals may
increase the amount or the antenna size of photosystem II
which would alter poising in favour of the production of
reducing equivalents.
When nitrate assimilation occurs in the dark in leaves,
or in roots, the reducing equivalents are provided by the
oxidative pentose phosphate pathway (OPPP), and the
reduced ferredoxin required by nitrite reductase is synthe-
sized from NADPH via ferredoxin-NADP-oxidoreduc-
tase. Nitrate addition leads to rapid induction of
ferredoxin (Matsumara et al. 1997) and ferredoxin-
NADP-oxidoreductase (Ritchie et al. 1994) in maize
roots. In the green algae Selenastrum minutum addition of
nitrate, but not ammonium, leads to a rapid activation of
glucose-6-phosphate dehydrogenase and a drop in glu-
cose-6-phosphate and an increase in 6-phosphogluconate
levels indicating that the OPPP has been stimulated
(Huppe, Vanlenberghe & Turpin 1992; Huppe & Turpin
1994; Turpin et al. 1997). The importance of cross-talk
between nitrate metabolism and the redox state of the cell
is underlined by the finding that NR expression (Mattana
et al. 1996) and activation (Botrel & Kaiser 1997)
increases in response to anaerobiosis.
3.5 Regulation of organic acid metabolism by
signals from nitrate and nitrogen metabolism
Assimilation of nitrate is accompanied by increased syn-
thesis of organic acids. 2-Oxoglutarate is required as an
acceptor in the GOGAT pathway, other organic acids are
required during the synthesis of other amino acids, and
malate is accumulated as a counter-anion to prevent alka-
linization during nitrate assimilation (Martinoia & Rentsch
1994; Heldt 1996). The synthesis of organic acids is regu-
lated at several levels in response to signals from nitrate,
ammonium and their metabolism.
Following addition of nitrate or ammonium to the alga
Selenastrum minutum, there are rapid changes in the con-
centrations of glutamine, glutamate, aspartate, 2-oxoglu-
tarate and malate that activate pyruvate kinase and PEP
carboxylase (PEPCase) and stimulate the flux of carbon
into organic acids (Huppe & Turpin 1994; Turpin et al.
1997). There is evidence for an analogous allosteric regula-
tion of these enzymes by organic acids and amino acids in
higher plants (Turpin et al. 1997).
Protein phosphorylation (see MacKintosh 1998; for a
recent review) allows a coordinate activation of PEPCase
(Foyer et al. 1994a; Duff & Chollet 1995; Li, Zhang &
Chollet 1996) and inhibition of sucrose phosphate synthase

(SPS) (Champigny et al. 1992; Manh et al. 1993; Huber &
Huber 1996) after adding nitrate or ammonium. Activation
of PEPCase is regulated via de novo synthesis of PEPCase-
kinase (Hartwell et al. 1996), resulting in decreased sensi-
tivity to inhibition by malate (Chollet, Vidal & O’Leary
1996). The malate sensitivity of PEPCase decreased in the
presence of nitrate in low-NR tobacco (Scheible et al.
1997b), indicating that nitrate-mediated signals contribute
to the post-translational regulation of PEPCase.
There is also evidence that signals from nitrogen
metabolism allow coordinate transcriptional regulation of
a group of key genes for organic acid synthesis. Nitrate
addition leads to an increase of the transcripts for
PEPCase, the cytosolic pyruvate kinase, citrate synthase
and the NADP-dependent isocitrate dehydrogenase
(NADP-ICDH) (Scheible et al. 1997b). These enzymes
convert phosphoenolpyruvate to 2-oxoglutarate which is
the primary carbon acceptor for inorganic nitrogen, and to
malate which acts as a counteranion during nitrate assimi-
lation. Crucially, these transcripts also change after adding
nitrate to transformants which have very low NR activity,
showing that the increase is triggered by nitrate itself rather
than other compounds that are formed during nitrate assim-
ilation (Scheible et al. 1997b). The transcript levels change
within 2–4 h, showing that nitrate acts rapidly and that high
internal concentrations are not required (Scheible et al.
1997b; and A. Krapp unpublished). Significantly, nitrate
addition does not lead to a significant increase of the tran-
script for fumarase, an enzyme in the section of the tricar-
boxylic acid cycle that is not required for net synthesis of
malate or 2-oxoglutarate (A. Krapp, unpublished results).
The changes in transcript levels were accompanied by an
increase of the activity of PEPCase (Foyer et al. 1994b;
Scheible et al. 1997b), pyruvate kinase, citrate synthase
and NADP-ICDH (A. Krapp, W.-R. Scheible and M. Stitt,
unpublished), and an accumulation of 2-oxoglutarate and
other organic acids (Scheible et al. 1997b).
Addition of nitrate or ammonium also leads to a rapid
and marked increase of the Ppc transcript and of PEPCase
activity in maize (Sugiharto et al. 1992b; Sugiharto &
Sugiyama 1992; Suzuki et al. 1994). Comparison of the
kinetics of various metabolite pools with the changes of the
Ppc transcript indicated in this case that glutamine may
play a key role in the induction of PEPcase. Addition of
nitrate or ammonium also led to an increase of PEPCase
activity in barley, which was prevented when phos-
phinothricin was added to inhibit GS activity (Diaz,
Lacuestra & Munoz-Rueda 1996), again indicating that
glutamine plays a key role in the induction of PEPCase in
this species.
3.6 Modulation of nitrate storage
The nitrate content of plant tissues varies enormously.
Nitrate levels are very low in nitrate-limited plants, and
increase when the nitrate supply is increased or the rate of
plant growth is decreased, for example in low light or short
days (Matt et al. 1998), or in transformants with low rates
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 593
of photosynthesis (Stitt & Schulze 1994). Most of the
nitrate is located in the vacuole (Miller & Smith 1996). It
has been suggested that this nitrate is not readily accessible
to metabolism. However, an interruption of the nitrate sup-
ply leads within hours to remobilization of the nitrate from
epidermal cells in the leaf and root (Dietz, Hollenbach &
Hellwege 1994; van de Leij et al. 1998). There are also
large (> 20-fold) changes in the nitrate pool in source
leaves during the day (see, e.g. Scheible et al. 1997c).
Microelectrode measurements have shown that the
cytosolic nitrate level is maintained remarkably constant,
irrespective of the level in the vacuole and of whether
nitrate is being accumulated or released (Miller & Smith
1996; van de Leij et al. 1998). These results imply that the
cytosolic nitrate concentration is tightly regulated, and that
the movement of nitrate into and out of the vacuole is not
related to changes in the cytosolic nitrate level (see discus-
sion in van de Leij et al. 1998).
The transport proteins involved in nitrate transport
across the tonoplast, in the entry of nitrate into the xylem,
and in the entry and release of nitrate from cells in the stem
and leaves have not yet been identified or cloned. Recently,
we have observed that nitrate is remobilized very rapidly
from the roots and stems of low-NR tobacco transfor-
mants, and that this remobilization can be markedly
slowed by supplying glutamine (A. Krapp, and M. Stitt,
unpublished results). These results indicate that down-
stream signals generated during nitrate assimilation regu-
late the remobilization of stored nitrate.
3.7 Regulation of the level of the AgpS transcript
and of starch accumulation by nutrients
Nitrate limitation typically leads to a large increase of the
starch content. It has been tacitly assumed that starch accu-
mulation is a passive response to the decreased rates of
growth, and the resulting accumulation of sugars. High
levels of sugars can lead to increased starch synthesis via
feedback regulation of the sucrose synthesis pathway,
resulting in an increase of the 3PGA:phosphate ratio and
allosteric activation of AGPase, the key regulatory enzyme
in the pathway of starch synthesis (Neuhaus & Stitt 1990;
Preiss 1991; Stitt 1991, 1996; Huber & Huber 1996).
Sugars also exert transcriptional control on AgpS (Müller-
Röber et al. 1990; Krapp & Stitt 1995; Koch 1996; Nielsen
et al. 1998), the gene encoding the regulatory subunit of
AGPase. However, recent research shows that nitrate, and
other nutrients including phosphate, are able to modulate
starch accumulation in a more direct manner.
The AgpS transcript level decreases within 2–4 h when
nitrate is added to nitrogen-deficient tobacco, and
increases again after removal or use of the nitrate (Scheible
et al. 1997b). Crucially, the same changes are seen after
adding nitrate to tobacco transformants with low NR activ-
ity, showing that the signal is related to nitrate itself rather
than the metabolization of nitrate (Scheible et al. 1997b).
Further, the rate of starch remobilization after adding
nitrate is similar in wild-type plants and in the low-NR
594 M. Stitt and A. Krapp
transformants, even though the rate of growth is not signif-
icantly altered in the latter (Scheible et al. 1997b). The
observation that the changes in starch metabolism can be
effectively uncoupled from the changes in growth rates and
carbohydrate consumption demonstrates the importance of
nitrate-related signalling for the regulation of starch
metabolism. Incidentally, this repression of AgpS is not due
to a general inhibition of carbohydrate synthesis by nitrate.
The Sps transcript and SPS activity are unaltered or even
increased after adding nitrate to wild-type plants or low-
NR transformants (Scheible et al. 1997b).
The AgpS transcript level also decreases when phosphate
is supplied to detached leaves (Nielsen et al. 1998).
Phosphate acts antagonistically to exogenous sugars, but
the effect of phosphate is not due to changes in the endoge-
nous sugar levels as these do not change after feeding
phosphate (Nielsen et al. 1998). It is not yet known if phos-
phate itself, or specific phosphorylated metabolites, trigger
the increase in the AgpS transcript.
Transcriptional regulation of starch metabolism in
response to signals that are derived from nutrients like
nitrate and phosphate will allow a feedforward regulation
of carbon allocation, and permit coordinate changes in car-
bohydrate allocation and nutrient assimilation and utiliza-
tion, without this requiring large intervening changes in the
sugar pools. In some cases, the direct effects of nutrients on
carbon allocation may be so powerful that they replace or
even override the regulation by sugars. This would explain
why nitrate- and phosphate-deficient plants sometimes
contain larger amounts of starch and lower levels of sugars
in their leaves than nutrient-sufficient plants (Brooks 1986;
Rao & Terry 1989; Fichtner et al. 1993; Scheible et al.
1997a; Geiger et al. 1999).
3.8 The sugar-mediated repression of genes
involved in photosynthesis is modulated by
nitrogen status
3.8.1 The nitrogen supply affects the levels of enzymes
involved in photosynthesis
When the nitrogen supply to a plant is decreased there is a
general decrease of protein and chlorophyll, but the rela-
tive amounts of Rubisco, thylakoid proteins and other leaf
proteins are not markedly affected (Evans 1996). In some
cases, there is a slight decrease of Rubisco relative to total
protein (Terashima & Evans 1988; Fichtner et al. 1993;
Makino et al. 1992, Makino, Nakana & Mae 1994) which
explains why Rubisco exerts more control over the ambi-
ent rate of photosynthesis in nitrogen-deficient than in
nitrogen-sufficient plants (Quick et al. 1993). This is in
striking contrast to changes in the growth irradiance, which
lead to marked shifts in the relative amounts of chlorophyll
and various proteins (Evans 1996).
Although it is hardly surprising that the overall protein
concentration and, more specifically, the amount of Rubisco
and other enzymes of photosynthesis increase when nitrogen
fertilizer is supplied, the molecular mechanisms underlying
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
this increase have not been investigated. It is unlikely that
the nitrogen-driven increase of overall protein is a purely
passive response (see Section 3.10). Intriguingly, low-NR
transformants growing on high nitrate contained slightly
higher Rubisco activity than nitrate-limited wild-type
tobacco, or the same transformants growing on limiting
nitrate (Lauerer 1996). Further, whereas the RbcS tran-
script level decreases dramatically in nitrate-deficient
wild-type tobacco, it remains significantly higher in low-
NR transformants when they are grown on high nitrate
(W.-R. Scheible, A. Krapp, M. Stitt, unpublished results).
3.8.2 Sugar-mediated regulation of RbcS expression is
modulated by the nutrient status
Removal of nitrogen leads to a cessation of growth and a
remobilization of protein from the source leaves. Recently,
Paul & Driscoll (1997) showed that the decline of leaf pro-
tein and Rubisco after removing nitrogen can be prevented
if the light intensity is simultaneously decreased, to pre-
vent carbohydrates from accumulating. This result indi-
cates that there is a close interaction between nitrogen- and
sugar-signalling events regulating leaf protein levels.
The interactions between nitrogen status and sugar-
mediated repression of RbcS have also been investigated
by growing tobacco plantlets in nutrient agar on different
ammonium nitrate levels in the presence and absence of
sucrose (Paul & Stitt 1993; Nielsen et al. 1998). Nitrogen-
limited plants contained lower amounts of total protein and
lower Rubisco activity, but the RbcS transcript level was
not decreased relative to total RNA. When sucrose was
supplied to nutrient-sufficient plants, growth was
increased, and protein, Rubisco activity and the RbcS tran-
script level remained high (see also Kovtun & Daie 1995).
When sucrose was supplied to nitrogen-limited plants,
there was a marked decrease of the RbcS transcript level,
Rubisco, protein and chlorophyll.
This increased susceptibility to sugar-repression in nitro-
gen-limited plants is not just a consequence of slow
growth, because sucrose did not lead to a decrease of pro-
tein, Rubisco activity or the RbcS transcript level in phos-
phate-limited plants (Nielsen et al. 1998). Indeed, in older
plants, phosphate limitation typically leads to an increase
of the activity of Rubisco and other enzymes that are
required during photosynthesis (Brooks et al. 1986; Rao &
Terry 1989; Fredeen et al. 1990). Low temperature also
leads to an increase of the leaf sugar content and an
increase of the activities of the Calvin cycle enzymes
(Holaday et al. 1992; Hurry et al. 1994, 1995), and Strand
et al. (1997) recently showed that low temperature leads to
an increase of the levels of the transcripts for RbcS and
other Calvin cycle enzymes in Arabidopsis leaves, even
though sugars simultaneously accumulate to high levels.
Taken together, these results indicate that sugars only
repress the genes for Calvin cycle enzymes in specific cir-
cumstances. There is a striking analogy with the effect of
elevated [CO2] on photosynthesis, Rubisco activity and the
RbcS transcript level in nitrogen-and phosphate-deficient

plants (see Riviere-Rolland et al. 1996; discussed in
Section 2.1). The resulting specificity may be important,
because it allows the acclimation response to be adjusted,
depending on the nutrient stress encountered. Thus,
decreased expression of the proteins for photosynthesis in
older leaves during nitrogen stress will allow reallocation
of nitrogen to support growth of roots, young leaves or
reproductive organs, whereas increased activity of the
Calvin cycle enzymes during phosphate stress will con-
tribute to the maintenance of photosynthetic rate, and
increased activity of the Calvin cycle enzymes as a result
of cold acclimation will also contribute to the maintenance
of photosynthetic rate, protect against photoinhibition,
and allow the accumulation of sugars that act as a cryopro-
tectant (see Strand et al. 1997 and Nielsen et al. 1998 for
further discussion).
3.9 Regulation of whole plant allocation, root
architecture and phenology by nitrogen signalling
Nitrogen fertilization not only leads to increased growth,
but also results in alterations in allocation and phenology
(see above). Recent experiments using transformants with
low NR activity have revealed that nitrate-mediated sig-
nalling triggers at least some of these changes in allocation
and development.
Plants growing with a low nitrogen supply typically
have a large root biomass relative to the shoot biomass
(Brouwer 1962; Van de Werf & Nagel 1996), and the
roots are often much more finely branched than in well-
fertilized plants (Grime et al. 1991; Fichtner & Schulze
1992). In low-NR tobacco transformants, however, the
accumulation of nitrate was accompanied by a strong
inhibition of root growth and stimulation of shoot growth,
resulting in shoot : root ratios that were higher than in
nitrogen-replete wild-type plants (Scheible et al. 1997a)
even though the low-NR transformants were severely
nitrogen-limited with respect to organic nitrogen. Split-
root experiments indicated that the inhibition of root
growth was triggered by the accumulation of nitrate in
the shoot, not the root. The inhibition of root growth was
correlated with decreased allocation of carbon to root
growth (Scheible et al. 1997a) and with a selective
decrease in the number of lateral roots (Stitt & Scheible
1998; Stitt and Feil 1999).
Similar results have also been obtained in Arabidopsis
(Zhang & Forde 1998; Meyer zu Hoerste 1998).
Interestingly, Lexa & Cheeseman (1997) did not find any
difference in the shoot-root ratio in a pea NR-deficient
mutant. This indicates that there may be species-depen-
dent differences in the extent to which nitrate and other
signals regulate shoot-root allocation. Significantly, pea
(Lexa & Cheeseman 1997) also differed from tobacco
(Scheible et al. 1997a) and Arabidopsis (Meyer zu
Hoerste 1998) in having relatively low levels of nitrate in
the leaves, in not showing such a marked increase of leaf
nitrate in response to a rising nitrate supply in the medium,
and in having lower nitrate in the shoot than the roots.
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 595
These differences might, speculatively, be linked to the
ability of pea to form root nodules, in which case nitrate
would not be the major nitrogen source.
The action of nitrate on root growth is complex, because
localized application of nitrate leads to a local stimulation
of lateral root proliferation (Drew & Saker 1975; Granato
& Raper 1989). Localized application of nitrate still leads
to a proliferation of lateral roots in tobacco (Scheible et al.
1997a) and Arabidopsis (Zhang & Forde 1998) genotypes
with very low NR activity, and the root proliferation is not
accompanied by an increase of the local concentrations of
amino acids or protein (Scheible et al. 1997a), showing
that it again involves nitrate-mediated signalling, rather
than a simple nutrient-mediated increase in growth. It will
be important in the future to investigate how the internal
nitrate pool and the external distribution in the soil interact,
to regulate the overall rate of root growth, the location of
root growth, and root architecture.
Further changes induced by nitrogen fertilization
include a delay in flowering, and a stimulation of germina-
tion (see above). The delay in flowering of Arabidopsis can
probably be attributed to the accumulation of nitrate, rather
than high levels of amino acids or protein (Meyer zu
Hoerste 1998). The induction of seed germination also
occurs independently of the assimilation of nitrate
(Hilhorst & Karssen 1989).
3.10 Molecular analysis of nitrogen signalling
Summarizing the last sections, nitrate triggers a coordi-
nated change in the expression and activity of a pro-
gramme of enzymes involved in nitrate and ammonium
assimilation, organic acid synthesis, and starch synthesis,
resulting in a redirection of metabolism away from starch
synthesis and towards nitrogen assimilation and amino
acid production, and also triggers changes in whole plant
allocation and in development that affect root growth and
architecture, flowering and seed germination. It is probable
that other compounds in nitrogen metabolism interact with
nitrate, for example glutamine antagonizes the nitrate-
mediated induction of the high affinity nitrate transporter,
NR and nitrite reductase, and is likely to reinforce the posi-
tive effects of nitrate on events lying further down-stream
in metabolism, allocation and growth. The molecular dis-
section of nitrate- and other nitrogen-signalling pathways
in plants is still in its infancy. The following section briefly
outlines the cardinal features of nitrogen-signalling path-
ways in other model organisms, and then reviews current
research on nitrogen signalling in higher plants.
3.10.1 Nitrogen sensing in bacteria
In Enterobacteria, nitrate acts to regulate two genetically
distinct different sets of NR and nitrite reductase genes that
have different functions (Stewart 1994). One pathway uses
nitrate as an electron acceptor in anaerobic conditions and is
induced by nitrate at low oxygen tension (Unden et al.
1995). Nitrate is sensed by two overlapping two-component
596 M. Stitt and A. Krapp
regulatory systems (Kaiser & Sawers 1995; Darwin &
Stewart 1995). The sensor components (NARX, NARQ)
interact with nitrate and nitrite and modify the activity of
the response elements (NARL, NARP), which are DNA-
binding proteins. NARX and NARQ both contain a con-
served histidine residue that is needed for the
phosphorylation and activation of the response regulators
NARL and NARP (Cavicchioli et al. 1995). NARX and
NARQ are thought to sense nitrate in the periplasmic
space (Cavicchioli et al. 1996; Chiang, Cavicchioli &
Gunsallis 1997).
The corresponding genes for a genetically and biochem-
ically distinct NR and nitrite reductase that function in the
pathway for nitrate assimilation are encoded on the Ntr
regulon in Escherichia coli. The feedback regulation of
nitrate assimilation by down-stream metabolites has been
intensively investigated. Transcription of the Ntr regulon is
inhibited when reduced nitrogen sources are available, via
a cascade which includes the bi-functional UTase/UR
(urididyl transferase/urididyl removing enzyme), the PII
protein, the bifunctional kinase/phosphatase NRII, and the
transcription factor NRI (Kamberov et al. 1994). The regu-
lation of this cascade by metabolites may involve measure-
ment of the glutamine concentration by the UTase/UR
protein, and of 2-oxoglutarate by the PII protein
(Kamberov, Atkinson & Ninfa 1995).
In Klebsiella pneumoniae a pathway-specific factor
NASR has been identified that mediates nitrate-induction
of the nitrate assimilation pathway (Goldman, Lin &
Stewart 1994; Lin & Stewart 1996). The leader sequence
of the operon contains a transcription terminator, and
NASR may act by overriding this termination signal (Lin
& Stewart 1996). Expression of the nitrate assimilation
pathway is also subject to general nitrogen control medi-
ated by the NTRC protein, which leads to repression when
ammonium is supplied (Goldman et al. 1994; Lin &
Stewart 1996).
3.10.2 Nitrogen sensing in fungi
A similar picture, in which of nitrate uptake and nitrate
assimilation are subject to a pathway-specific induction by
nitrate, and are repressed by ammonium and products of
ammonium as part of a general response to these metabo-
lites, is also found in fungi. The best studied systems are
Aspergilus nidulans and Neurospora crassa (Crawford &
Arst 1993; Marzluf 1993a, 1993b). Nitrate induction of
Nit3 (encoding NR) is mediated by the pathway-specific
regulatory protein NIT4 in Neurospora, and the homolog
NIRA in Aspergillus. Ammonium repression is mediated by
the globally acting regulatory protein NIT2 in Neurospora
and the homolog AREA in Aspergillus, whereby NIT2 and
AREA act positively to increase the expression the enzymes
for nitrate assimilation and a large number of unlinked
structural genes that specify nitrogen-catabolic enzymes in
conditions of nitrogen limitation, specifically low glu-
tamine. Operation of NIT2 is regulated by a negative-acting
regulator NMR (Xiao, Fu & Marzluf 1995).
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
3.10.3 Nitrogen sensing in eukaryotic algae
A NIT2 homolog has been cloned from Chlamydomonas,
where it is required for expression of three nitrate and
nitrite transporter proteins, NR, and nitrite reductase,
which form a gene cluster reminiscent of that found in
fungi (Galvan, Quesada & Fernandez 1996). Nit2 is
expressed in the presence of nitrate and is repressed by
ammonium (Quesada et al. 1993; Schnell & Lefebvre
1993). Two loci, Nrg1 and Nrg2 have been identified that
are required for the ammonium-repression of the nitrate
transporters, NR and nitrite reductase activity, although
they may act in a different manner to the NIT4/NIRA sys-
tem in fungi (Prieto et al. 1996). There is evidence that NR
itself regulates the expression of the nitrate transport pro-
teins (Navarro et al. 1996).
NR is also subject to post-transcriptional and post-
translational regulation in Chlamydomonas, although the
mechanisms differ form those in higher plants. As in
tobacco species, constitutive overexpression of Nia leads
to an increase of the Nia transcript but not necessarily of
NR protein. In Chlamydomonas, nitrate appears essential
for the increase of active NR protein (Navarro et al.
1996). Activation of NR may be related to a redox inter-
conversion process (Franco, Cardenas & Fernandez
1987). As in higher plants, there is evidence that inactiva-
tion of NR acts as a signal for its subsequent degradation
(Franco et al. 1987).
3.10.4 Nitrogen sensing in plants
Nitrogen sensing in higher plants is likely to be even more
complex than in micro-organisms. Plants are multicellular
organisms, and nitrogen affects many facets of metabolism
and physiology at the cellular and the whole plant level.
There are two further intriguing differences between plants
and micro-organisms. First, due to the prevalence of nitrate
in the soil and the problems of pH regulation that arise dur-
ing ammonium assimilation, most species that have been
investigated in detail appear to utilize nitrate as their major
and preferred nitrogen source. Second, the large changes in
glutamine that can occur in leaves as a consequence of
photorespiratory ammonium release may complicate the
use of glutamine as a general signal for the nitrogen status
of the plant, at least in leaf mesophyll cells.
The analysis of nitrogen sensing in higher plants has
been seriously hampered because the types of screen (e.g.
insensitivity to chlorate, Nia expression mutants) that
yielded large numbers of regulatory mutants in other
organisms did not yield regulatory mutants or have proved
refractory in higher plants. This may be due to the com-
plexity of the regulation networks and/or the presence of
redundant regulatory proteins. The identification of a large
number of other genes whose expression is modulated by
nitrate or by other nitrogen metabolites (see above,
Sections 3.1–3.5) and the identification of nitrate-modu-
lated processes that provide visual phenotypes (Section
3.7) could open new possibilities to isolate mutants in

nitrogen-signalling processes in the near future. In addi-
tion, several other approaches have started to yield infor-
mation about nitrogen-signalling processes in plants.
One involves the use of sequence homologies or func-
tional complementation screens to isolate putative
homologs to genes that are involved in nitrogen signalling
in other organisms. A NIT2 homolog (Daniel-Vedele &
Caboche 1993) and an Arabidopsis PII homolog that lacks
the uridenylation site (G. Coruzzi, personal communica-
tion) have been identified in plants. A functional comple-
mentation screen using a yeast mutant deficient in Gln3 (a
yeast homolog of AreA and Nit2) has allowed the isolation
of two genes designated Rga1 and Rga2 (Truong et al.
1997), which are members of a multigene family that
includes SCARECROW which is involved in pattern for-
mation in roots (Di Laurenzio et al. 1996), and have subse-
quently turned out to be identical with Gai (Peng et al.
1997) and Rga (Silverstone, Ciampaglio & Sun 1998)
which are implicated in giberellin signal transduction.
In a second approach, Zhang & Forde (1998) have used
differential screening to identify genes that are rapidly
induced after addition of nitrate, and isolated a novel
nitrate-regulated gene in Arabidopsis (Anr1) which
encodes a member of the MADS-box family of transcrip-
tion factors. Antisense inhibition of Anr1 expression pre-
vents proliferation of lateral roots in response to local
application of nitrate, so identifying ANR1 as a important
component of the signalling pathway by which nitrate
modulates lateral root growth.
In a third approach, Sakakibara et al. (1998) have pro-
vided evidence that a cytokinin-inducible protein in maize
leaves (pZm CIP1) is involved in the signal transduction
pathways of nitrogen signalling. Earlier results from this
group had already shown that the C4-cycle PEPCase can
be induced by nitrogen sources in intact plants but not in
detached leaves where, instead, cytokinins are effective in
increasing PEPCase expression (Sugiharto, Burnell &
Sugiyama 1992a; Suzuki et al. 1994). The level of the pZ
mCip1 transcript and pZmCIP1 protein also increase tran-
siently after supplying nitrate or ammonium to the roots of
maize plants, but not after supplying nitrate or ammonium
to detached leaves (Sakakibara et al. 1998). Further,
isopentyladenosine accumulates in the root within 2 h of
adding nitrate (Sakakibara et al. 1998). Taken together,
these results suggest a model for a nitrogen-sensing system
which involves the synthesis of cytokinins in the roots, fol-
lowed by translocation of the cytokinins to the shoot and
induction of pZ mCip1 in the leaf, although the precise role
of pZm CIP1 still has to be established. Intriguingly, the
sequence of pZ mCip1 shows homologies to the response
regulator element of bacterial two component signalling
systems. A small family of response regulator proteins
(ARR3-ARR7) that show a similar cytokinin and nitrate-
dependent expression has recently been identified in
Arabidopsis (Taniguchi et al. 1998).
These results are fascinating because cytokinins are
already implicated in other nitrogen-mediated events.
There is indirect evidence implicating cytokinins in the
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 597
increase of the level of the Nia transcript level and NR
activity after adding nitrate to barley plants (Samuelson,
Campbell & Larsson 1995), and zeatin riboside has been
implicated in the regulation of NR in Chenopodium cell
suspension cells (Peters, Füchbauer & Beck 1995). The
formation of lateral roots is known to be induced by auxin
and inhibited by cytokinins (Webster & Radin 1972).
Nitrogen fertilization leads to increased levels of
cytokinins in the roots and root sap (Kuiper et al. 1989;
Samuelson, Eliason & Larsson 1992; Wagner & Beck
1993; Sakakibara et al. 1998), and the effects of nitrate
fertilization on shoot-root allocation can be mimicked by
adding cytokinin (Fetene & Beck 1993). Further evidence
for an interaction between nitrogen signalling and
cytokinins is provided by the phenotype of a low nitrogen-
sensitive mutant isolated by Faure, Jullien & Caboche
(1994). This mutant maps to the Zea3 complementation
group that confers resistance to zeatin, and exhibits a
light-dependent sensitivity of cotyledon growth to a low
nitrogen/carbon ratio.
It will also be important to identify which nitrogen
pools or processes in nitrogen metabolism are sensed. As
outlined in Section 3.8, there is evidence that shoot nitrate
rather than root nitrate may be involved in the regulation
of shoot-root allocation in tobacco. The finding that the
cytosolic nitrate level is fairly constant (see Section 3.6)
implies that sensing systems for nitrate may be located in
the apoplast or at the plasmalemma, and in the vacuole or
at the tonoplast. The observation that the Nia transcript
level responds to re-irrigation with nitrate, even when the
leaves still contain a large pool of nitrate (Geiger et al.
1998b; D. Klein and M. Stitt, unpublished) provides inde-
pendent evidence that the inflow rather than the internal
pool of nitrate is being sensed.
With respect to glutamine signalling, an intriguing
problem arises in leaves, because ammonium and glu-
tamine are produced during photorespiration as well as
during de novo nitrate and ammonium assimilation. The
major pools of ammonium and glutamine appear to be
shared by both processes (Scheible et al. 1997c; Geiger
et al. 1998b), which raises interesting problems with
respect to the extent to which signals derived from these
pools can give specific information about the nitrogen
status. The cytosolic GS1 occurs as cell specific isoforms,
including one expressed specifically in the phloem com-
plex (Dubois et al. 1996; Lam et al. 1996). Since GS1
expression responds more specifically to changes in sug-
ars and nitrate (see Sections 3.3 and 4.3), and there may
be important cell-specific events that are not seen in over-
all measurements of metabolites.
Summarizing, although the molecular analysis of nitro-
gen signalling in higher plants is still in its infancy, the
information available paints a tantalizing picture in which
signals derived from nitrate and nitrogen metabolism inter-
act with other fundamental processes in the regulation of
plant gene expression, plant hormones and plant develop-
ment. These interactions underline their potential impor-
tance for the control of plant metabolism and development.
598 M. Stitt and A. Krapp

4 NITROGEN METABOLISM IS REGULATED nitrate uptake has been provided by studies of the diurnal
BY SIGNALS THAT ARE DERIVED FROM changes of nitrate uptake in soybean plants (Delhon et al.
CARBON METABOLISM 1995). Nitrate uptake is faster in the light period than the
dark period, and evidence for a role of sugars was provided
The potential contribution of sugar-mediated regulation of
by three different manipulations. This light-dependent stim-
gene expression for understanding the acclimation of pho-
ulation was prevented by removal of carbon dioxide from
tosynthesis was pointed out in Stitt (1991). Since then
the atmosphere on the previous day or girdling of the stem to
sugar-mediated signalling has been implicated in the regu-
prevent sugar export to the roots, and was restored by pro-
lation of a host of further processes (Koch 1996; Graham
viding sucrose to the roots. Further, the rate of nitrate uptake
1996; Smeekens & Rook 1997), including nitrogen
during the night was decreased even further by decreasing
metabolism (Hoff et al. 1994; Krapp & Stitt 1995; Lam
the light intensity during the preceding light period to reduce
et al. 1996). The following section discusses how nitrate
starch accumulation and, thence, the supply of sucrose dur-
uptake, assimilation and utilization can be regulated in
ing the ensuing dark period. Recently, it has been observed
response to signals derived from carbon metabolism. The
that addition of sugars to the rooting medium leads to a two-
term ‘sugar-mediated’ regulation is used, without implying
fold higher levels of the Nrt2 : 1 transcript in N. plumbagini-
a particular mechanism whereby the sugars are leading to
folia seedlings (A. Krapp, unpublished).
the observed changes in transcript levels or metabolism
Nevertheless, plants growing in low light intensities or
that result after feeding sugars (see Section 4.5 for a dis-
in short days (Matt et al. 1998) and transformants with low
cussion of the mechanisms of sugar sensing).
rates of photosynthesis due to antisense inhibition of the
There are two overriding reasons why nitrogen
expression of Calvin cycle enzymes (Stitt & Schulze 1994;
metabolism needs to be regulated in response to changes in
V. Haake and M. Stitt, unpublished) typically contain
carbon metabolism. Firstly, the utilization of sucrose in
extremely high levels of nitrate. This indicates that nitrate
growing sinks will normally depend upon simultaneous
uptake is inhibited less than nitrate assimilation when car-
provision of suitable amounts of amino acids. Secondly,
bohydrates are low. The energetic demands of nitrate
nitrate assimilation depends on and in some conditions
uptake are much lower than the energetic and carbon costs
even competes with carbon metabolism. Nitrate assimila-
for nitrate assimilation, and a large pool of nitrate would
tion diverts large amounts of carbon away from carbohy-
provide a reserve of nitrogen that can be immediately
drates and towards the synthesis of organic acids (see
exploited if conditions change and photosynthesis and
Section 3.5). The assimilation of nitrate and the synthesis
growth can be increased.
of amino acids requires reducing equivalents and ATP
(Section 3.4), and therefore competes with photosynthetic
carbon fixation and carbohydrate synthesis in leaves in 4.2 Nitrate assimilation
limiting light, and requires catabolism of carbohydrates to
Whereas nitrate can be readily stored and can even perform
provide ATP and reducing equivalents in leaves in the dark
an important function as an osmoticum, nitrite is toxic, and
and in non-photosynthetic tissues. Over 20% of the total
ammonium may also be toxic at high concentrations.
root respiration may be related to nitrate uptake and assim-
Further, since the protonated forms are volatile, when nitrite
ilation in barley roots (Bloom, Sukrapanna & Warner
and ammonium accumulate to high concentrations they may
1992). Provided carbohydrates are not limiting, the carbon
exit the plant, which would represent a considerable loss of
required for nitrate assimilation can be obtained by reduc-
energy and nitrogen. For these reasons, NR represents a key
ing the rate of starch accumulation or by re-mobilizing
site where the flow of nitrogen into organic compounds is
starch or sugar reserves that were accumulated at an earlier
regulated in response to the supply of carbohydrate.
time point (Waring et al. 1985; Fichtner & Schulze 1992;
Scheible et al. 1997a). However, when carbohydrates are
in short supply, continued nitrate assimilation could lead to 4.2.1 Transcriptional regulation
an metabolic imbalance, resulting in accumulation of
Sugars are implicated in the transcriptional regulation of
ammonium, a shortfall of organic acids to counteract acidi-
Nia in leaves. The Nia transcript level increases in
fication, or a lack of carbon for export or respiration.
detached leaves after feeding sugars, especially when the
Conversely, when carbohydrates are available in large
leaves have been predarkened to deplete the endogenous
amounts, it will be important to increase the rate of nitro-
sugars (Cheng et al. 1992; Vincentz et al. 1993), and in
gen assimilation.
leaves on intact plants when the petiole is cooled to inhibit
phloem export and increase the endogenous sugar levels
(Krapp & Stitt 1995). Sugars are also implicated in the reg-
4.1. Nitrate uptake
ulation of Nia expression in roots. During prolonged dark-
There have been relatively few studies of the effects of ening, the level of the Nia transcript decreases, and this is
sugar supply on nitrate uptake, and the effects of changes reversed when sugars are supplied to the roots (Botrel &
in carbohydrate levels on the expression of the various Kaiser 1997; Sivasankar, Rothstein & Oaks 1997).
nitrate transport proteins has not yet been investigated. It is less clear, however, whether and when sugar-medi-
Physiological evidence that the sugar supply influences ated signals make a major contribution to the regulation of
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621

NR assimilation. Correlations between changes of sugars
and the Nia transcript level or NR activation in leaves dur-
ing light–dark transitions and diurnal rhythms cannot be
unambiguously interpreted, because many other potential
regulators including nitrate and glutamine are changing
simultaneously. The evidence for an effect of sugars on Nia
transcript and NR activity is mainly based on feeding
experiments in which rather high levels of sugars were
supplied exogenously (Vincentz et al. 1993; Sivasankar et
al. 1997; Botrel & Kaiser 1997). The levels of sugars that
accumulate after petiole cooling are much higher than are
normally found in leaves (Krapp & Stitt 1995) and,
although NR activity decreased dramatically in antisense
RbcS transformants, this decrease was only found in lines
with a large reduction of Rubisco activity, photosynthesis
and sugar levels (Stitt & Schulze 1994). When tobacco
plants are grown in short day-conditions or exposed to an
extended night, Nia transcript levels and NR activity do not
decrease until sugars are below about 3–4 µmol/g FW
(Matt et al. 1999). Recently, we investigated the effect of
transferring low-NR tobacco transformants to permanent
darkness (D. Klein, A. Krapp. M. Stitt, unpublished
results). The Nia transcript level did not decrease for 2 to
3 d, by which time the sugar content had fallen to very low
levels. These studies all indicate that sugar-mediated regu-
lation only plays a major role in the regulation of Nia tran-
scription when the sugar levels in leaves are very low. The
observation that sucrose and glutamine act antagonistically
on the level of the Nia transcript level (Morcuende et al.
1998), however, suggests that higher levels of sugars may
be required in conditions where the plants are less depleted
in amino acids.
4.2.2 Post-translational regulation of NR
Sugars are also implicated in the post-translational regula-
tion (see Section 3.2.2) of NR. Dark-inactivation is
reversed if sugars are supplied to leaves in the dark (Kaiser
& Brendle-Behnisch 1991; Kaiser & Huber 1994a), and
light-activation does not occur if carbon fixation is pre-
vented by water stress or low [CO2] concentrations (Kaiser
& Förster 1989; Kaiser & Brendle-Behnisch 1991).
Experiments with extended night treatments indicate that
post-translational regulation of NR is sensitive to relatively
small changes of sugar levels (Matt et al. 1998; D. Klein,
A. Krapp and M. Stitt, unpublished results). As discussed
in Section 3.2.3, there is evidence that the rate of NR
degradation may also be initiated by NR phosphorylation,
in which case sugar-mediated regulation may contribute to
the regulation of NR protein turnover.
4.3 Ammonium uptake and assimilation
The cytosolic glutamine synthetase (GS1) transcript level
increases in the light, and this increase can be mimicked by
adding sugars in the dark (Lam et al. 1996). It is not known
if this leads to changes in GS1 protein, nor whether post-
translational mechanisms or allosteric effectors regulate
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 599
GS activity. Recently, Morcuende et al. (1998) showed in
feeding experiments with detached leaves that sugars lead
within 2 to 4 h to an increased flux from nitrate into glu-
tamine and other amino acids, even though the levels of
glutamate and ammonium decrease, providing strong evi-
dence that sugars stimulate GS activity in vivo.
4.4 Provision of carbon skeletons for ammonium
assimilation, and amino acid synthesis
The subsequent synthesis of amino acids depends on car-
bon skeletons produced by photosynthesis and sugar
catabolism. Relatively little is known about the regulation
of organic acid synthesis and amino acid synthesis by sug-
ars. However, some recent experiments indicate that the
stimulation of de novo nitrogen assimilation is accompa-
nied by a coordinated regulation of carbon flow into the
pools of organic acids and from there into the amino acid
biosynthesis pathways.
4.4.1 Synthesis of organic acids
Recent experiments indicate that the sugar-mediated stim-
ulation of nitrate and ammonium assimilation described in
Sections 4.2 and 4.3 is accompanied by a coordinated stim-
ulation of organic acid synthesis. Morcuende et al. (1998)
showed that sucrose stimulates the synthesis of organic
acids in detached tobacco leaves. Irrespective of whether
the sucrose was supplied on its own, or with nitrate, or with
glutamine, it led to a marked increase of 2-oxoglutarate, a
small decrease of isocitrate, and a large decrease of the gly-
colytic precursors glycerate-3-phosphate (3PGA) and
phosphoenolpyruvate (PEP). These results show that
organic acid synthesis is being stimulated via activation of
specific steps, including those catalysed by PEPCase,
pyruvate kinase and NADP-isocitrate dehydrogenase, and
not just as a passive response to an increase supply of sub-
strates. These changes were seen within 2 to 4 h, indicating
that the sugars are leading to rapid changes in the activity
of these enzymes. Leport et al. (1996) reported that dark-
ening or exposure to CO2 free-air led to an inhibition of in
vivo PEPCase activity as monitored by 14C fixation,
directly showing that changes in photosynthetic carbohy-
drate production lead to rapid changes in PEPcase activity.
The increased rates of dark fixation found by Leport et al.
(1996) were not accompanied by a change in the in vitro
activity or a marked change in the malate sensitivity of
PEPcase, indicating that the activation may involve
allosteric regulation rather than protein phosphorylation.
Although PEPCase can be regulated by protein phosphory-
lation (see Section 3.5), the contribution during sugar-
mediated activation has not yet been established.
Sugar feeding also leads to an increase of the levels of
the transcripts for several important enzymes involved in
the synthesis of organic acids (Koch 1996), including the
cytosolic pyruvate kinase (Krapp et al. 1993) and the
NADP-dependent isocitrate dehydrogenase (NADP-
ICDH) (Fieuw et al. 1995). However, it is not yet known
600 M. Stitt and A. Krapp
whether the sugar-mediated increase of the transcript levels
leads to an increase of the encoded proteins. Rapid diurnal
changes in the transcripts for PEPCase, cytosolic pyruvate
kinase and NADP-ICDH and two- to three-fold changes in
the activities of the encoded enzymes have recently been
observed in tobacco leaves (A.Krapp, W.-R Scheible and
M. Stitt, unpublished results).
4.4.2. Synthesis of the minor amino acids
The minor amino acids (the aromatic amino acids,
branched and unbranched chain aliphatic amino acids,
lysine, histidine, arginine, cysteine, methionine, proline)
are synthesized from a variety of carbon precursors, using
glutamate, aspartate and, occasionally, glutamine as the
amino donor. The regulation of amino acid synthesis by
feedback regulation from the individual amino acid end
products has been extensively researched (Lea & Forde
1994). Amino acid synthesis in mammals and fungi is also
regulated by the general nitrogen control system, in which
GNC4 acts as a transcription factor to promote transcrip-
tion of more than 30 amino acid biosynthesis genes in
response to starvation for a single amino acid (Dever et al.
1992; Pain 1994). There is evidence that Opaque-2 is a
functional plant homolog of GNC4 (Mauri et al. 1993)
although its function in processes other than seed storage
protein synthesis has not been investigated in detail.
Relatively little is known about the regulation of amino
acid biosynthesis by the availability of the carbon precur-
sors. Carbon starvation leads to a general decrease of
amino acids in a sugar cane suspension culture (Veith &
Komor 1993). This could be due to an inhibition of amino
acid synthesis, or a stimulation of amino acid catabolism.
Evidence that prolonged carbon starvation leads to amino
acid catabolism is provided by the finding that asparagine
synthetase is induced when leaves are darkened (Lam et al.
1996) and during starvation of detached maize root tips
(Brouquisse et al. 1992; Chevalier et al. 1996), and is
repressed again if sugars are added. Matt et al. (1998)
showed that a transient decrease of sugars after a single
dark night leads to a rapid and reversible decrease of all the
minor amino acids and an increase of glutamate and aspar-
tate, and that re-illumination leads to an increase of the
minor amino acids and a simultaneous decrease of gluta-
mate and aspartate. Further, Morcuende et al. (1998)
showed that when sugars are supplied to detached source
leaves the rates of nitrate assimilation and the levels of all
the minor amino acids increase, but the levels of glutamate
and aspartate decrease. These results indicate that sugars,
directly or indirectly, stimulate flux into the amino acid
biosynthesis. It will be important in the future to investi-
gate whether sugars stimulate the expression and activity
of enzymes in amino acid biosynthesis pathways.
Summarizing the previous sections, there is mounting
evidence that sugar-mediated signalling modulates nitro-
gen metabolism, acting at several sites including nitrate
assimilation itself, ammonium assimilation, the synthesis
of organic acids, and possibly the flow of nitrogen and
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
carbon into the various amino acid biosynthesis pathways.
These sugar-mediated signals will complement the nitro-
gen-mediated signals discussed in Section 3. In agreement,
it is necessary to provide nitrate and sucrose to achieve
maximum rates of nitrate assimilation and amino acid syn-
thesis in detached leaves (Morcuende et al. 1998).
4.5 Mechanisms of sugar sensing
The mechanisms involved in sugar sensing in plants
have been recently reviewed (Sheen 1994; Thomas et al.
1995; Koch 1996; Graham 1996; Jang & Sheen 1997;
Smeekens & Rook 1997; Smeekens, 1998). There is
evidence that hexokinase may play an important role as a
glucose sensor in plants (Jang & Sheen 1994; Jang et al.
1997), as has also been suggested in yeast. It is, however,
likely that the mechanisms involved in sugar sensing in
plants will be considerably more complex than in yeast.
Plants are multicellular organisms, and contain different
sorts of cells with strikingly different requirements for
and dependence on sugars.
The evidence for the role of hexokinase as a sugar-sen-
sor is partly based on experiments with glucose analogues
like mannose and 2-deoxyglucose to show that phosphory-
lation is a requirement for the signal but further metabo-
lization is not required (Sheen 1994; Jang & Sheen 1994;
Martin et al. 1997; Koch 1996), The use of glucose ana-
logues is, however, compromised because there is consid-
erable metabolization of the phosphorylated derivative in
plant cells (Klein & Stitt 1998). The other stronger line of
evidence involves the analysis of the effect of glucose on
gene expression transformants with decreased expression
of hexokinase and, especially, transformants with overex-
pression of ectotopic hexokinases (Jang et al. 1997).
However, the way in which hexokinase functions as a
sugar sensor has not yet been elucidated at the molecular
level, even in yeast (Smeekens, 1998).
Some evidence is apparently inconsistent with the opera-
tion of hexokinase as a glucose sensor in plants. Tobacco
transformants with overexpression of yeast invertase
showed strikingly different phenotypes, depending upon
whether the invertase was overexpressed in the apoplast, the
vacuole or the cytosol (Sonnewald et al. 1991). Whereas the
apoplast- and vacuole-overexpressors showed a marked
bleaching, a loss of chlorophyll (Sonnewald et al. 1991),
decreased protein and Rubisco levels (M. Brauer and M.
Stitt, unpublished results), decreased levels of the Cab tran-
script, and induction of wound-induced proteins (Herbers
et al. 1996), transformants with overexpression of invertase
in the cytosol did not show changes in expression of cab and
wound-induced proteins (Herbers et al. 1996) nor a decrease
of Rubisco activity (M. Brauer and M. Stitt, unpublished
results). Herbers et al. (1996) proposed that hexose sensing
occurs in the endomembrane lumen. An alternative explana-
tion (Smeekens, 1998) would be that sensing requires move-
ment of hexoses back into the cytoplasm from either the
apoplast of the vacuole, which would still be compatible
with a role for hexokinase but would require the additional

postulate that it only acts as a sensor in association with
specific transport processes.
The picture is further complicated by recent evidence
from yeast that sugar transporter homologs can function as
hexose sensors to regulate a subclass of the sugar-mediated
responses (Özcan et al. 1996). There is already evidence that
the transport of hexoses rather than their metabolism acts as
the signal to induce some groups of sugar-responsive genes
in plants. Thus, transportable but non-metabolizable glucose
analogs like 6-deoxyglucose were able to induce invertase
and sucrose synthase in Chenopodium cells (Godt, Riegel &
Roitsch 1995; Roitsch, Büttner & Godt 1995) and reporter
genes linked to the sugar-responsive potato patatin-1 promo-
tor in Arabidopsis (Martin et al. 1997).
Other recent evidence indicates that plants can also sense
sucrose. Sucrose was more effective than reducing sugars in
inducing patatin expression in detached potato leaves
(Wenzler et al. 1989; Jefferson, Goldsbrough & Bevan
1990), a rolC-promotor GUS reporter gene construct was
activated more effectively by sucrose feeding than by glu-
cose or fructose in the phloem of tobacco seedlings
(Yokoyama et al. 1994), and sucrose was more effective
than glucose in decreasing sucrose symporter activity and
transcript levels in detached leaves (Chiou & Bush 1998).
Sucrose was also more effective than glucose in activating
nitrate assimilation and the synthesis of organic acids in
tobacco leaves (Morcuende et al. 1998). Recently, it has
been shown that sucrose represses synthesis of a protein
termed ATB2, probably acting at the level of translation, and
that glucose or fructose are less effective (Rook et al. 1998).
ATB2 is a vascular-tissue specific BZIP transcription factor,
and might therefore play a role in mediating sucrose-
induced changes in gene expression in sink tissues. It should
be noted however, that a rapid cycle between sucrose and
hexose sugars in many plant tissues (see Dancer, Hatzfeld &
Stitt 1990; Goldschmidt & Huber 1992; Krapp et al. 1993;
Geigenberger & Stitt 1993) will complicate the interpreta-
tion of experiments where different sugars are supplied. It is
also important to include a control in which similar amounts
of glucose or fructose, i.e. double the molarity of the sucrose
concentration added, are included and this has not always
been done in studies to date that report a selective effect of
sucrose on gene expression.
There is mounting evidence that sucrose and reducing
sugars exert a differing influence on sink metabolism and
development. Invertase activities are high in stolons but fol-
lowing tuberization, the activity of invertase decreases and
sucrose synthase increases, with the result that growing
tubers contain relatively low levels of hexose sugars and
high levels of sucrose (Oparka et al. 1992; Ross et al. 1994;
Appeldoorn et al. 1997). Potato tuber-specific overexpres-
sion of yeast invertase leads to a decrease of the starch con-
tent and a stimulation of respiration (Sonnewald et al.
1997), and these changes are accentuated when glucokinase
is introduced into the invertase-overexpressing tubers, even
though the concentrations of all of the glycolytic precursors
are massively increased (Trethewey et al. 1998). Further,
addition of glucose promotes respiration in potato discs,
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 601
whereas sucrose promotes starch accumulation even though
the levels of the glycolytic metabolites decrease (Geiger
Stitt & Geigenberger 1998a). In an elegant analysis of gene
expression and carbohydrate profiles in developing Vicia
seeds, Wobus and coworkers have shown that high inver-
tase activity and high levels of hexoses are found in zones
characterized by cell division, whereas starch accumulation
is found in zones characterized by high sucrose synthase
and high sucrose (Heim et al. 1993; Weber et al. 1995a,
1995b, 1997a, Weber, Borisjuk & Wobus 1997b; Borisjuk
et al. 1998).
It is therefore important to avoid premature generaliza-
tions about the mechanisms whereby sugars regulate gene
expression and metabolism in plants. Sugar feeding or
endogenous accumulation of sugars lead to far-reaching
changes in many different aspects of plant metabolism and
development, including processes related to rapid
catabolism, cell division, wounding, photosynthesis, nitrate
assimilation and storage processes. Some of these processes
are linked or share common features, but some are mutually
antagonistic, and different sugars or sensing mechanisms
may be involved. This means that caution is needed in
assuming that the molecular details of the sensing mecha-
nism that are revealed by analysis of the sugar-mediated reg-
ulation of one set of genes in one tissue and physiological or
developmental state are relevant for other tissues, states, or
groups of genes. Caution is also needed in assuming that
effects produced by feeding sugars are caused by the sugars
themselves, rather than the metabolization of the sugars, or
even more indirect effects due to changes in other processes
that result as an indirect consequence of sugar-feeding.
An important recent development has been the isolation
of mutants with altered sugar-sensing properties
(Smeekens & Rook 1997). The plants becoming available
include rsr mutants altered in the susceptibility of the
patatin-1 promotor to sugar-induction (Martin et al. 1997),
sun mutants showing decreased susceptibility to sugar
repression of a plastocyanin promotor-LUC reporter gene
construct in tobacco seedlings (Dijkwel et al. 1997), and
mutants altered in the sugar-induction of (α – amylase
(Mita et al. 1997). These mutants will provide an invalu-
able resource to analyse the mechanisms of sugar sensing,
to define subclasses of responses, and to investigate if the
nitrogen supply or elevated [CO2] affect any, some, or all
of these responses. In addition, they are already providing
strong evidence for cross-talk between sugar sensing and
other signal transduction pathways. SUN6 and SUN7 mod-
ulate two specific subclasses of PHYA responses in a sugar
dependent manner (Dijkwel et al. 1997).
5 THE SIGNIFICANCE OF
CARBON : NITROGEN INTERACTIONS FOR
UNDERSTANDING AND ANALYSING THE
RESPONSE OF METABOLISM AND GROWTH
TO ELEVATED CARBON DIOXIDE
As mentioned in Section 2, although numerous studies
have described the interaction between elevated [CO2] and
602 M. Stitt and A. Krapp
nitrogen supply, few of these studies included a detailed
analysis of nitrogen metabolism. A detailed analysis of
nitrogen metabolism is required to understand the mecha-
nisms that govern the response to elevated [CO2], for two
reasons. Firstly, increased rates of plant growth in elevated
[CO2] may lead to plants becoming nitrogen limited or
exacerbate an existing nitrogen limitation, and some of the
changes in metabolism and physiology in elevated [CO2]
might be indirect effects, due to the plants becoming more
nitrogen limited. It will therefore be important to analyse
nitrogen metabolism in parallel with changes in photosyn-
thesis, carbon metabolism and growth to identify condi-
tions in which complicating effects due to changes in the
nitrogen status are absent or minimized, and to distinguish
between direct effects of elevated [CO2] and an increased
supply of photosynthate, and indirect effects due to nitro-
gen limitation. Secondly, a detailed analysis of nitrogen
metabolism is needed to identify changes in specific pools
in nitrogen metabolism which might be of significance for
the regulation of metabolism, development and growth in
elevated [CO2]. This could include factors which increase
nitrogen uptake and assimilation, or the allocation of nitro-
gen in the cell and the plant.
5.1 Evaluation of the nitrogen status in elevated
carbon dioxide
Obviously, correct interpretation of experiments with ele-
vated [CO2] will depend on an adequate set of criteria to
assess whether the plant nitrogen status has been signifi-
cantly changed. This has traditionally been done by measur-
ing the total nitrogen content, and in some cases by resolving
total nitrogen into nitrate and total organic nitrogen, or mea-
suring total protein (see Section 2.4). Such measurements
may be useful when they can be interpreted against a large
background of empirical information for a particular species
and type of location, but have several serious limitations if
this background information is lacking.
The interpretation of changes of total leaf nitrogen is
complicated by the large amounts of nitrate that can accu-
mulate in well-fertilized plants. Due to the dramatic diur-
nal changes in the leaf nitrate content (Gebauer, Melzer &
Rehder 1984; Scheible et al. 1997c), the nitrate content
measured at an arbitrary time of the day will not give reli-
able information about the nitrogen status of the plant.
Further, the nitrate present in a plant leaf at a given time in
its life history represents a surplus, that has not been
reduced and used to produce organic nitrogen. To interpret
the functional significance of a change in the nitrate pool,
harvests at several time points during the growth of the
plant will be required to reveal whether or not the nitrate is
subsequently utilized. If the nitrate is not later remobilized,
a decrease of nitrate in elevated [CO2] would have little or
no functional significance for plant growth, although it
may affect the nutritional quality of the litter. The amount
of nitrate accumulated can anyway vary greatly between
species (se Section 3.1.1).
Changes of total organic nitrogen or protein are also
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
difficult to assess on their own because they are not large
(see Poorter et al. 1997) and because Rubisco itself repre-
sents 20 to 50% of leaf protein (Evans 1989; Fichtner
et al. 1993; Makino et al. 1997). Based on changes of
total protein, it is not possible to distinguish between (i) a
decrease of leaf protein due to selective decrease of
Rubisco and possibly other proteins involved in photo-
synthesis, and (ii) a general decrease of leaf protein.
These two potential explanations, however, will have
very different functional consequences (see also the dis-
cussion in Baxter et al. 1997).
To avoid the complication caused by the special case of
Rubisco, Baxter et al. (1997) suggested that the root pro-
tein content is a better marker for the plant nitrogen status.
They also proposed further useful parameters which can be
monitored to detect changes in the plant nitrogen status
including an alteration in the shoot : root ratio and prema-
ture senescence of older leaves. A further useful criteria is
to use a range of nitrogen supplies including concentra-
tions that are shown to be super-optimal for plant growth.
Another very powerful approach would be to compare the
changes of total leaf protein with the changes of Rubisco
and a group of other selected photosynthesis proteins and
of a group of housekeeping proteins.
It is also advisable to carry out a detailed analysis of the
levels of the various nitrogen metabolites in the plant.
There have been remarkably few investigations of the
effect of elevated [CO2] on the levels (Ferrario-Mery et al.
1997; Geiger et al. 1998b) and the diurnal changes (Geiger
et al. 1998b) of nitrate, ammonium and the individual
amino acids. As discussed in Section 3.4, nitrogen-suffi-
cient plants show highly dynamic changes in the pools of
nitrogen metabolites. During the light period nitrate
decreases, and considerable amounts of ammonium and of
selected amino acids accumulate, especially glutamine,
glycine and serine. These changes are then reversed during
the night. (Ferrario-Mery et al. 1997; Geiger et al. 1998b).
Many of the other amino acids, including the minor amino
acids, do not show such marked changes (Geiger et al.
1998b). Further, the effect of the nitrogen supply on the
levels of nitrate, ammonium and selected amino acids such
as glutamine and asparagine or on the glutamine : gluta-
mate or asparagine : aspartate ratios are far faster and, in
relative terms, far larger than the changes of total nitrogen,
total protein, Rubisco, chlorophyll or total amino acids
(Geiger et al. 1998b; Geiger et al. 1999). They therefore
provide better information about short- and long-term
changes in the plant nitrogen status, and will also provide
an early warning that a nitrogen limitation is developing,
long before significant changes in source leaf protein or the
overall nitrogen concentration are detectable. Several of
these parameters including nitrate, glutamine and
asparagine may also act as internal signals for the current
nitrogen status (see Section 3). It may also be useful to
complement measurements of leaf pools with measure-
ments of the pools in the roots, where the diurnal changes
are less marked (Scheible et al. 1997c; Geiger et al.
1998b).

Studies that have included a detailed analysis of the
changes in nitrogen metabolism in elevated [CO2] provide
the following picture. (i) In plants receiving a super-opti-
mal nitrogen supply, elevated [CO2] either leads to an
increase of nitrate (Ferrario-Mery et al. 1997; Geiger et al.
1998b) or, even when it leads to a decrease (Geiger et al.
1999), the nitrate level is still high. The overall level of
amino acids, the levels of most of the individual amino
acids (Ferrario-Mery et al. 1997; Geiger et al. 1998b,
1999), and the protein content (Baxter et al. 1997; Geiger
et al. 1998b) in the leaves and the roots do not decrease sig-
nificantly, and may even show a small increase. Although
there is a decrease of ammonium and of selected amino
acids including glycine, serine and glutamine (Ferrario-
Mery et al. 1997; Geiger et al. 1998b) this related to
changes in photorespiration (see below) rather than to
changes in the nitrogen status of the plant. (ii) When ele-
vated [CO2] is supplied to nitrogen-limited plants, there is
a relatively small decrease of leaf and root protein
(Ferrario-Mery et al. 1997; Baxter et al. 1997; Geiger et al.
1999), a decrease of nitrate (Ferrario-Mery et al. 1997;
Geiger et al. 1999), and a decrease of leaf amino acids
which includes a particularly marked decrease of glu-
tamine, glycine and serine (Ferrario-Mery et al. 1997;
Geiger et al. 1999). There is a decrease of total root nitro-
gen (Baxter et al. 1997), which includes a decrease of the
amino acids and protein (Geiger et al. 1999). (iii) The dif-
ference between plants growing in ambient and elevated
[CO2] is especially marked when the nitrogen supply is
just sufficient for nitrogen-sufficient growth in ambient
[CO2], because elevated [CO2] leads to the plants becom-
ing nitrogen limited. This is accompanied by large changes
of nitrate, ammonium and glutamine, and a marked
decrease of protein and a wide range of enzyme activities
in the leaf (Geiger et al. 1999). It will be difficult to inter-
pret the effect of elevated [CO2] on metabolic or physio-
logical parameters in intermediate nitrogen regimes,
because many of the observed changes may just be indirect
effects caused by the change in the nitrogen status. By
careful choice of the fertilization regime (see below for
further discussion), however, it may be possible to mini-
mize these effects.
5.2 The regulation of nitrogen metabolism in
elevated carbon dioxide
As discussed in the last section, exposure of suboptimally
fertilized plants to elevated [CO2] results in the appearance
or accentuation of a nitrogen limitation. This could be due
to a combination of factors, including exhaustion of the
available nitrogen in the rooting medium, decreased avail-
ability of the nitrogen in the soil (see also Section 2.6), and
dilution due to rapid growth and a more rapid progression
through the life cycle (see Baxter et al. 1997). On the other
hand, when plants which are receiving a super-optimal
supply of inorganic nitrogen are exposed to elevated
[CO2], they are able to increase the nitrogen uptake and
assimilation to keep pace with or even overtake the
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 603
increased rate of carbon fixation and growth. The follow-
ing section will relate recent investigations of nitrogen
metabolism in elevated [CO2] to the emerging information
about the role of sugars and nitrogen metabolites in the reg-
ulation of nitrogen metabolism (Sections 3 and 4).
5.2.1 Uptake of nitrate and ammonium
The reported effects of elevated [CO2] on nitrate and
ammonium uptake are extremely variable (Section 2.6).
The overall rate of nitrate uptake can be increased in
elevated [CO2], resulting in an increased concentration of
total nitrogen and the major components of the nitrogen
household plants growing in high nitrate in hydroponics
and in young seedlings (see Section 5.1). The failure to see
a stimulation of nitrate uptake in other systems could be
due to a variety of factors, including depletion of soil
nitrate which is needed to induce nitrate transporters (see
Section 3.2). The molecular cloning of the high and low
affinity nitrate transporters and the ammonium transporters
will enable the effect of elevated [CO2] on the expression
of the various transporters to be studied, and compared
with the changes of nitrate, ammonium and nitrogen
metabolites. It will also allow transformants to be pro-
duced with altered expression of the transporters, to test
whether nitrate or ammonium uptake is a limiting step for
growth in elevated [CO2].
5.2.2 Nitrate assimilation
The reported effects of elevated [CO2] on NR activity are
also very variable (Section 2.7). Nonetheless, elevated
[CO2] clearly leads to increased NR activity in Plantago
(Fonseca et al. 1997) and tobacco (Geiger et al. 1998b).
The reasons for this increase can be quite complex. In
tobacco, elevated [CO2] did not markedly increase NR
activity in the first part of the light period when NR activity
is at its diurnal peak, but it decreased or prevented the
decline of NR activity in the second part of the photope-
riod, and partially reversed the dark-inactivation of NR
(Geiger et al. 1998b). It also led to a change in the diurnal
regulation of NR in roots (Geiger et al. 1998b).
Although there was a slight and transient increase of the
Nia transcript level after transferring Plantago major to
elevated [CO2] (Fonseca et al. 1997), prolonged growth in
elevated [CO2] led to a slight decrease of the Nia transcript
level in N. plumbaginifolia (Ferrario-Mery et al. 1997) and
tobacco (Geiger et al. 1998b) leaves. This decrease was
seen in nitrogen-sufficient plants (Ferrario-Mery et al.
1997; Geiger et al. 1998b) even in seedlings, where the
internal pools of nitrate and the current growth rate were
increased in elevated [CO2] (Geiger et al. 1998b). Elevated
[CO2] also fails to prevent the rapid decay of the Nia tran-
script during the photoperiod (Scheible et al. 1997c;
Geiger et al. 1998b). The absence of a positive effect of
elevated [CO2] on the Nia transcript level is consistent
with the evidence (see Section 4.2.1) that plants growing in
ambient [CO2] contain enough sugars for them not to be a
604 M. Stitt and A. Krapp
major factor in regulating the level of the Nia transcript
level, except when the light regime is unfavourable for
photosynthesis.
Two lines of evidence indicate that elevated [CO2] acts,
instead, via post-transcriptional and post-translational
mechanisms to increase NR activity. First, elevated [CO2]
reduced the decline of NR activity and protein during the
second part of the photoperiod (see Section 3.2.1), and par-
tially reversed the dark inactivation (see Section 3.2.2) of
NR (Scheible et al. 1997c; Geiger et al. 1998b). Second,
when N. plumbaginifolia transformants expressing Nia
under the control of the constitutive 35S promotor were
compared with wild-type plants in hydroponic culture on
high nitrate, NR activity was only 30% higher in the trans-
formants in ambient [CO2] whereas it was 100% higher in
elevated [CO2] (Ferrario-Mery et al. 1997). Interestingly,
this stimulatory effect of elevated [CO2] on NR expression
in the 35S transformants was reversed when the genotypes
were compared in pots with a lower nitrogen supply. This
indicates that uncharacterized post-transcriptional mecha-
nisms also contribute to the decrease of NR activity in ele-
vated [CO2] when nitrogen becomes limiting.
The increased activation and increased stability of NR in
elevated [CO2] might be a result (see Section 4.2.2) of the
higher levels of sugars in elevated [CO2] (see Section 2.2).
However, an intriguing similarity between the diurnal
changes of NR protein and NR activation in elevated
[CO2] (see above) and the changes in tobacco mutants with
a decreased number of functional Nia gene copies
(Scheible et al. 1997c) suggests an alternative explanation.
In both cases, the decay of NR protein during the photope-
riod was decreased, dark-inactivation was partially
reversed and, crucially, these changes correlated with a
slower accumulation of glutamine during the light period.
These results indicate that the altered diurnal regulation of
NR in elevated [CO2] may be partly due to a weakening of
the feedback regulation exerted by glutamine or related
metabolites that are formed during nitrate assimilation.
The lower level of glutamine in elevated [CO2] may be
partly due to increased export of glutamine or increased
conversion of glutamine to other amino acids as a result of
the higher sugar levels (see Section 4.4). It may also (see
Section 5.2.3) be partly due to the decreased rate of pho-
torespiration in elevated [CO2].
Elevated [CO2] also leads to increased NR activity in the
roots (Fonseca et al. 1997; Geiger et al. 1998b). The
increased supply of sugars may play an important part in
the regulation of root NR expression and activity. Fonseca
et al. (1997) observed an increase of the Nia transcript
level and a larger and more sustained increase of root NR
activity than shoot NR activity after transferring Plantago
major plants to elevated [CO2], and suggested that this was
due to the increased availability of sugars in the root.
Geiger et al. (1998b) grew tobacco plants for several
weeks in elevated [CO2], and found that their roots con-
tained higher NR activity than in ambient [CO2] as a result
of a change in the diurnal rhythm of root NR activity. A
transient increase of sugars at the start of the photoperiod
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
was followed by an increase of NR activity, which in turn
was followed by a decrease of nitrate and an increase of
nitrite and amino acids, and then by a decrease of sugars to
lower levels than in ambient [CO2], and a decrease of NR
activity (Geiger et al. 1998b). The decrease of sugars might
be due to their consumption during nitrate assimilation.
Two studies have shown that the extent and timing of the
increase in NR activity in elevated [CO2] correlates with
the time period during which the current growth rate is
stimulated. After transfer of Plantago major to elevated
[CO2], growth was only stimulated during the first days,
and this growth stimulation was not accompanied by a
decrease of reduced nitrogen (Fonseca et al. 1997). When
tobacco was grown continuously in elevated [CO2], the rel-
ative growth rate was only stimulated between 12 and 20 d
compared to that in ambient [CO2], and at this time the lev-
els of amino acids and protein were higher in elevated than
in ambient [CO2] (Geiger et al. 1998b). In both cases, it
was proposed that the up-regulation of nitrate assimilation
plays an important role in allowing higher rates of growth
in elevated [CO2].
Several factors could explain why many other studies
have found unaltered or even decreased NR activity in ele-
vated [CO2] (see Section 2.7). Firstly, in tobacco the
increase of NR activity was due to a change in the diurnal
regulation (see above), and studies investigating activity at
a single time point might not detect these subtle changes.
Secondly, elevated [CO2] could lead to a preferential assim-
ilation of ammonium, resulting in formation of glutamine
and (see Section 3.3) repression and/or post-translational
inactivation and degradation of NR. There have been very
few comparisons of the response to elevated [CO2] in the
presence of nitrate and ammonium nitrate, and many stud-
ies have used a mixture of nitrate and ammonium fertiliza-
tion. Geiger et al. (1999) recently observed that elevated
[CO2] always led to a 30 to 50% decrease of NR activity in
leaves of plants growing on super-optimal ammonium
nitrate, whereas it leads to an increase of NR activity in
plants growing on super-optimal nitrate. Thirdly, the low
NR activity in some studies in elevated [CO2] might be due
to the plants having become nitrate-deficient. NR activity
decreases when plants are nitrate-limited, because nitrate is
an important inducer of Nia expression (see Section 3.3).
5.2.3 Ammonium assimilation
As discussed in Section 3.2.2, any discussion of ammo-
nium assimilation is complicated by the massive recycling
of ammonium during photorespiration. This is especially
so in the case of elevated [CO2], when there will be a
change in the rate of photorespiration. An increase of the
external [CO2] from 0·035 to 0·07 Pa will approximately
halve the rate of photorespiration (Stitt 1991), releasing
substantial GS capacity for de novo assimilation of ammo-
nium, especially since (see Section 2.7) overall GS activity
does not decline in the leaves in elevated [CO2].
The decrease of photorespiration explains the substantial
decreases in the pools of ammonium, glycine and serine in

elevated [CO2] (Ferrario-Mery et al. 1997; Geiger et al.
1998b). This decrease is found, irrespective of whether the
plants are receiving nitrate or ammonium nitrate (Geiger
et al. 1999), and irrespective of whether they are nitrogen
limited or nitrogen sufficient (Ferrario-Mery et al. 1997;
Geiger et al. 1999). The decreased gross flux through the
GOGAT pathway could be one of the reasons why elevated
[CO2] led to a slower accumulation of glutamine during
the photoperiod, even in well-fertilized plants (Geiger
et al. 1998b). A decrease of glutamine could have consider-
able consequences for the regulation of metabolism and
growth in elevated [CO2] because, as discussed in Section
3.10, glutamine is one of the compounds that is sensed to
provide information about the nitrogen status. The possible
consequences for the regulation of NR in elevated [CO2]
was discussed in Section 5.2.2. Changes in the concentra-
tions of ammonium and glutamine in the leaves might also
lead to altered translocation of amino acids to the roots and
affect the expression of transporters (see Section 3.1), and
could have more far-reaching effects if glutamine turns out
to play a central role in nitrogen signalling as it does in
other organisms (see Section 3.10).
5.2.4 Amino acid synthesis
Elevated [CO2] only led to a small increase in the levels of
the minor amino acids during the light period, but it led to a
two-fold increase of almost all the minor amino acids in
tobacco leaves at the end of the night, even though gluta-
mate and aspartate were not significantly increased
(Geiger et al. 1998b). This might be explained by the
increased activity of NR activity during the night in ele-
vated [CO2] (Section 5.2.1) and by the stimulatory effect of
sugars on the synthesis of amino acids (Section 4.4).
Further studies are needed to investigate the consequences
for the rate of protein synthesis, and the synthesis of sec-
ondary metabolites that derive from various amino acids.
5.2.5 Nitrogen allocation
Changes in nitrogen allocation occur at two very different
levels in plants: at a cellular level due to changes in the dis-
tribution of nitrogen between the various proteins and
other nitrogen-containing compounds, and at a whole plant
level due to changes in the allocation of nitrogen to differ-
ent organs. Although the changes in nitrogen allocation in
elevated [CO2] have been frequently described (see
Sections 2.1, 2.4, also Section 5.3.1) and clearly depend on
the nitrogen status of the cell and the plant (see Sections
2.1, 2.3, 3, 5.3.1), the molecular basis for these changes is
not understood. At the cellular level, this will require a bet-
ter understanding how the expression of individual genes
and programmes of genes is regulated by sugar- and nitro-
gen-mediated signals (see Sections 3 and 4). At the whole
plant level, it will depend upon identifying the processes
that initiate and control the rate of growth of different
sinks, and regulate their entry into senescence. The identi-
fication of a large family of amino acid transporters
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 605
(Fischer et al. 1998; Rentsch, Boorer & Frommer 1998)
that show differences in their expression between differ-
ent tissues and cells and during development (Hirner et al.
1998) could provide a valuable tool to probe the mechanistic
basis for changes in nitrogen allocation. The growing aware-
ness of the interactions between nitrogen and sugar sensing
and hormones (see Sections 3.10 and 4.5) opens a comple-
mentary route to investigate how allocation is regulated at
the whole plant level (see below for further discussion).
5.3 Acclimation of photosynthesis
Several proximal explanations have been advanced for the
acclimation of photosynthesis to elevated [CO2], including
an inhibition due to damage caused by large starch grains,
a direct limitation of photosynthesis due to an inadequate
rate of end-product synthesis, and an inhibition due to a
decline of Rubisco and other proteins involved in photo-
synthesis (reviewed in Stitt 1991). As discussed in Section
2.1, a decrease of Rubisco has been observed in very many
studies of acclimation. This decrease could, however, have
different causes, including sugar-mediated signalling, an
interaction between sugar- and nitrogen-mediated sig-
nalling, or an indirect effect due to nitrogen limitation lead-
ing to earlier senescence. These will have very different
functional implications for the response of plants to ele-
vated [CO2], and the following section will discuss
whether it is possible to distinguish between these various
explanations for the data published to date.
5.3.1 A more complete data base will be required to
evaluate the reasons for the decrease in Rubisco
expression in elevated carbon dioxide
The decline of Rubisco activity or Rubisco protein in ele-
vated [CO2] is accompanied by a decrease of the tran-
scripts that encode RbcS and other transcripts encoding
proteins involved in photosynthesis (see Section 2.1).
Similar changes occur when sugars accumulate in leaves as
a result of an inhibition of phloem transport in transgenic
plants (Stitt 1991; Krapp et al. 1993; Riesmeier, Willmitzer
& Frommer 1994; Lerchl et al. 1995; Geigenberger et al.
1996), petiole cooling (Krapp & Stitt 1995), or sugar feed-
ing (Krapp, Quick & Stitt 1991; Paul & Stitt 1993; van
Oosten & Besford 1995; Nielsen et al. 1998). This similar-
ity prompted the hypothesis that the acclimation of photo-
synthesis in elevated [CO2] might be due to a
sugar-mediated repression of RbcS and other genes that are
required for photosynthesis (Stitt 1991).
Based on present models for sugar sensing in plants (but
see Section 4.5 for a word of caution), it has been proposed
that increased levels of hexose sugars lead, via hexokinase-
related signalling, to repression of RbcS and other genes
encoding proteins involved in photosynthesis, resulting in
a decrease of Rubisco and the other encoded proteins and
an inhibition of photosynthesis (Jang & Sheen 1994; Van
Oosten et al. 1995). In this model, the rate of recycling of
hexoses would be more important than the absolute
606 M. Stitt and A. Krapp
amounts of sugars and, particularly, the amount of sucrose
in the leaf. This would explain why the changes of the
RbcS transcript level and Rubisco protein and activity do
not correlate well with the absolute levels of sugars, either
after adding exogenous sugars (see, e.g. Krapp et al. 1993)
or during growth in elevated [CO2] (see Moore et al.
1999). Goldschmidt & Huber (1992) showed that there is a
correlation between acid invertase activity and the suscep-
tibility of photosynthesis to a ‘sink’-inhibition when sugars
accumulate after girdling. Recently, Moore et al. (1998)
showed in a study of 16 different species that elevated
[CO2] leads to a decrease of Rubisco protein in 11 species
where acid invertase activity exceeded a threshold value,
but only in one of the five species with low invertase activ-
ity. This is an attractive model that would involve a specific
sugar-sensing step in the acclimation to elevated [CO2].
However, there are still some serious deficiencies in the
experimental support for this model, and it is unlikely to be
the only explanation for photosynthetic acclimation.
Firstly, most of the experiments that reported a decrease
of Rubisco during photosynthetic acclimation were proba-
bly carried out with plants where elevated [CO2] led to the
appearance or the exacerbation of a nitrogen limitation (see
Sections 2.3 and 5.1). In the absence of direct evidence that
the plants did not become nitrogen-limited, it is difficult to
distinguish whether the changes in Rubisco activity or pro-
tein and the changes of transcript in a particular experiment
are triggered by sugar signalling, or by nitrogen limitation,
or by an earlier onset of senescence as a consequence of
nitrogen limitation, or other factors including self-shading
or ontogenetic drift. For this reason, most of the experi-
mental evidence for a decrease of the RbcS transcript and
Rubisco activity in elevated [CO2] is irrelevant to the dis-
cussion of the above model. It is particularly worrying that
elevated [CO2] did not lead to a decreased level of tran-
scripts and the encoded proteins or enzymes in several
studies where the nitrogen status was deliberately manipu-
lated or monitored, and the plants were nitrogen sufficient
(Riviere-Rolland et al. 1996; Habash et al. 1995; Atkinson
et al. 1997; Ludewig et al. 1998; Geiger et al. 1999).
In a study of wheat growing in elevated [CO2] in a FACE
system, Nie et al. (1995a) concluded that the decrease of
Rubisco activity in wheat leaves in elevated [CO2] might
have two phases: an early and small decrease which was
independent of major changes in chlorophyll and other
proteins including LHCII and the thylakoid ATP synthase,
and a later and large decrease which was accompanied by a
more general loss of protein and occurred in ambient
[CO2] but was accentuated in elevated [CO2], and might
reflect general leaf senescence which is speeded up in ele-
vated [CO2]. In a companion study, Nie et al. (1995b)
noted that elevated [CO2] resulted in a relatively large
increase of sugars and a relatively small decrease of the
levels of the transcripts for RbcS, RbcL and three other
Calvin cycle enzymes in young plants, and that in older
plants the difference became smaller with respect to sugar
levels but larger with respect to the transcript levels, again
indicating that further factors may be involved in the
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
decrease of these transcripts in elevated [CO2] in the older
plants. In Scirpus olneyi (Jacob et al. 1995) and another
study on wheat (Sicher & Bunce 1997), the decrease of
Rubisco and of total leaf protein were of the same order,
indicating that the decrease was due to a non-specific
decrease of leaf protein. In tobacco (Geiger et al. 1999),
elevated [CO2] had no effect on the levels of the transcripts
for RbcS, Cab, Rca (Rubisco activase) and NADP-glycer-
aldehyd-3-phosphate dehydrogenase or on the activity of
Rubisco and six other Calvin cycle enzymes or on the over-
all leaf protein content in nitrogen-replete plants grown on
10 mM ammonium nitrate or 20 mM nitrate, whereas it led
to a decrease of the levels of these four transcripts, a 20 to
30% decrease of Rubisco and six other Calvin cycle
enzymes, and a similar decrease of total leaf protein when
the plants were growing at a nitrogen supply (3 mM ammo-
nium nitrate or 6 mM nitrate) that was just adequate to
avoid nitrogen limitation in ambient [CO2] but became
limiting in elevated [CO2].
Secondly, several studies have shown that although ele-
vated [CO2] has a marked effect on Rubisco protein or
activity in older leaves, it does not lead to a decrease of
Rubisco in young leaves (Van Oosten et al. 1995; Nie et al.
1995a; Pearson & Brooks 1995; Miller et al. 1997). Miller
et al. (1997) have suggested that elevated [CO2] leads to a
shift in the timing of the normal photosynthetic stages of
leaf ontogeny, resulting in an earlier onset of the photosyn-
thetic decline.
Leaf senescence is a complex process that is regulated
by ontogenetic factors, and by many environmental factors
including the local light regime and the nitrogen supply
(Gan & Amasino 1997). There is probably a complex inter-
action between the environmental factors and the endoge-
nous developmental programme. This will make it difficult
to distinguish, on the basis of whole plant parameters,
between changes caused by a faster progression through
the life cycle per se, and changes induced by changes in the
nitrogen status or other external factors. This problem is
underlined by the complexity of the sugar- and nitrogen-
signalling systems (see Sections 3 and 4) and the emerging
evidence that they may interact with hormonal- and phy-
tochrome-signalling (see Sections 3.10 and 4.5). It will be
important to examine the leaf age-dependence of changes
in photosynthesis and Rubisco in parallel with an analysis
of the nutrient status and of genes that are specifically
induced during leaf senescence to show whether the devel-
opment of the plant has been speeded up in elevated [CO2],
or whether the earlier photosynthetic decline is triggered
by nitrogen deficiency.
Thirdly, even if photosynthetic acclimation is not always
just a consequence of earlier senescence or accelerated
ontogeny, the glucose-sensing model still does not explain
why the decrease of Rubisco is more pronounced in nitro-
gen-limited plants (see Section 2.1 and above). This discrep-
ancy cannot be explained on the basis of species variability.
In tobacco transformants with inhibited phloem function and
in petiole girdled tobacco leaves, accumulation of sugars
resulted in visual bleaching, a decrease of the RbcS tran-

script level, and a decrease of Rubisco, even though the
plants were not nitrogen-limited (see above for references).
In contrast, elevated [CO2] does not lead to a decrease of
the RbcS transcript or Rubisco activity in nitrogen-suffi-
cient tobacco, even though tobacco has high acid invertase
activity (Moore et al. 1998) and there is a substantial accu-
mulation of sucrose and hexoses in these conditions
(Geiger et al. 1999). Elevated [CO2] does not lead to a
decrease of Rubisco activity in well-fertilized wheat
(Habash et al. 1995) and oak (Atkinson et al. 1997), even
though the former also has high invertase activity (Moore
et al. 1999) and both species show a decrease of Rubisco
when less nitrogen is supplied (see, e.g. Nie et al. 1995b;
George et al. 1996). It is therefore necessary to postulate
either that signals derived from nitrogen metabolism mod-
ulate sugar sensing, or that there is cross-talk between the
sugar- and nitrogen-signalling pathways.
Fourthly, Moore et al. (1998) found in their species
comparison that the extent of the decrease of Rubisco pro-
tein was not correlated with the extent of the decrease of
the RbcS transcript. Species with modest reductions in
Rubisco (10–20%) often showed a marked reduction of the
RbcS transcript level, whereas species with a larger reduc-
tion in Rubisco (> 30%) only showed a small decrease of
the RbcS transcript level. This indicates that other factors
may be leading to the decrease of Rubisco in these species
that showed marked acclimation of photosynthesis.
These discrepancies might, of course, be due to difficul-
ties in obtaining material at a comparable stage in the accli-
mation process from a wide range of species. Interpretation
of transcript levels measured at a single time point may also
be problematic, because transcript levels can vary diurnally
and elevated [CO2] may modify these diurnal rhythms.
Recently, Cheng et al. (1998) showed that elevated [CO2]
indeed modifies the diurnal changes of the RbcS transcript
level in Arabidopsis, having little effect on the amount of
transcript during the day but leading to lower transcript lev-
els during the night. In tobacco growing in low nitrogen,
elevated [CO2] led to a decrease of the RbcS transcript level
that was first apparent as a decrease at the end of the light
period (Geiger et al. 1999). It should be noted, however,
that these complications will also weaken arguments that
are based on finding an apparent correlation between tran-
script levels that have been measured at a single time point
and changes in Rubisco levels and acclimation.
5.3.2 End-product synthesis may sometimes colimit
photosynthesis in plants growing in elevated carbon
dioxide
Stitt (1991) concluded from a literature review of studies
published up to that time that the accumulation of end
products was not responsible for the acclimation of photo-
synthesis. However, as discussed already, many earlier
studies are compromised by an interaction with nutrient
deficiency. The role of a direct end-product limitation
deserves re-examination in experiments where a possible
interaction with nitrogen supply is explicitly addressed.
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 607
One proposed mechanism invokes an accumulation of
starch grains leading to disruption of the plastids. In a
recent study on Pinus palustris, Pritchard et al. (1997)
showed that starch accumulation and plastid disruption is
especially marked when elevated [CO2] is combined with
low nitrogen. However, when potato transformants with
antisense inhibition of AGPase expression where grown in
elevated [CO2] at a high nitrogen supply they showed a
more marked acclimation of photosynthesis than wild-type
plants, showing that at least in this case, starch grains were
not directly causing the inhibition of photosynthesis
(Ludewig et al. 1998).
An alternative mechanism for a direct end-product inhi-
bition of photosynthesis is based on experiments on iso-
lated chloroplasts, and experiments on leaves subjected to
various treatments to sequester phosphate (Walker & Sivak
1986). In this model, low rates of end-product synthesis are
proposed to lead to an acute depletion of phosphate, a
decrease of ATP, an inhibition of 3PGA reduction, an
increase of 3PGA and deactivation of Rubisco (see Stitt
1991 for references). In a literature survey, Stitt (1991)
concluded that there was no convincing evidence for a
direct end-product limitation of photosynthesis when sug-
ars accumulate in leaves during prolonged exposure to ele-
vated [CO2], or as a result of other treatments that alter the
sink–source balance. Numerous investigations have found
that sugar accumulation in leaves, irrespective of whether
it is the result of sugar-feeding or an inhibition of phloem
transport or growth at elevated [CO2], is accompanied by
an increase of ATP, a decrease of 3PGA and increased acti-
vation of Rubisco (see also Krapp & Stitt 1995;
Morcuende et al. 1996, Morcuende, Pérez & Martinez-
Carrasco 1997; Morcuende et al. 1998 for more recent
publications). This is the opposite of the predicted response
if the rate of phosphate recycling during sucrose and starch
synthesis is colimiting for photosynthesis (see above).
Further, as also pointed out by Sage (1994), the changes in
the leaf gas exchange characteristics in plants showing
acclimation following long-term exposure to elevated
[CO2] are indicative of a decreased carboxylation capacity
rather than an inadequate rate of end-product synthesis.
For two reasons, however, this question needs to be re-
evaluated. First, the frequently observed changes in
carboxylation capacity are probably at least partly due to
changes in the nitrogen status (see Section 5.3.1). Second,
some recent results indicate that when sugars accumulate
in intact unstressed leaves, end-product limitation of
photosynthesis might generate a different metabolite pro-
file to that predicted on the basis of studies of isolated
chloroplasts or the effects of phosphate-sequestering
compounds on leaf metabolism. Morcuende et al. (1998)
showed that sugars strongly stimulate nitrogen metabolism
and organic acid synthesis (see Section 4.3), and the result-
ing stimulation of the respiratory pathways which will lead
to a decrease of the levels of 3PGA and other glycolytic
intermediates, and increased formation of ATP. There is a
pressing need for a re-examination of the role of end-
product limitation in plants growing in elevated [CO2] that
608 M. Stitt and A. Krapp
are demonstrably not subject to changes in their nitrogen
status. The growth temperature will need to be carefully
chosen and might affect the outcome of the experiments,
because the susceptibility of photosynthesis to inhibition
by Pi recycling increases at lower temperature (Stitt 1991,
1996).
Two subsequent studies of gas exchange characteristics
have shown that end-product limitation indeed makes a
contribution to the control of photosynthesis in elevated
[CO2] in some conditions and species. The oxygen-sensi-
tivity of photosynthesis (an indicator for a limitation of
photosynthesis by the rate of Pi recycling in sucrose and
starch synthesis, see Stitt 1991 for further discussion) was
decreased when bean (Socias, Medrano & Sharkey 1993)
and tobacco (Micallef et al. 1995) were grown at elevated
[CO2]. Acclimation of photosynthesis in elevated [CO2] in
bean was associated with a decrease of SPS activity and
decreased carbamylation of Rubisco, whereas Rubisco
protein was not reduced (Socias et al. 1993).
Studies of three different transgenic plants with altered
expression of enzymes required for sucrose or starch syn-
thesis have also provided evidence that inadequate rates of
end-product synthesis can contribute to acclimation. First,
tomato transformants with constitutive overexpression of
maize SPS and an increased capacity for sucrose synthesis
did not show a loss of the oxygen-sensitivity of photosyn-
thesis when they were grown in elevated [CO2], and did not
show an acclimation of photosynthesis to elevated [CO2]
(Micallef et al. 1995). One transformant line showed
increased biomass in elevated [CO2], compared to wild-
type plants (Leport et al. 1996). Galtier et al. (1995) also
found that overexpression of maize SPS led to a larger stim-
ulation of photosynthesis during a short-term exposure to
elevated [CO2]. Second, potato transformants with
decreased expression of AGPase and, hence, a decreased
capacity for starch synthesis showed a large and faster acc-
climation to elevated [CO2] than wild-type plants (Ludewig
et al. 1998). Significantly, acclimation was not accompa-
nied by a decrease of the RcbS or Cab transcript levels, or
Rubisco activity. Third, antisense inhibition of plastid
aldolase expression led to a stronger inhibition of photosyn-
thesis in plants growing in elevated [CO2] than in plants
growing in ambient [CO2]. This inhibition was accompa-
nied by a dramatic decrease of starch, an accumulation of
sugars, and a high 3PGA:triose phosphate ratio, and the
inhibition was not accompanied by a preferential decrease
of the transcripts for RbcS and other Calvin cycle enzymes,
nor was there a preferential decrease of the corresponding
enzyme activities (V. Haake, M. Geiger, R. Zrenner, P. Liu-
Walsch, C. Engels and M. Stitt, unpublished).
Summarizing, although there are several potential expla-
nations for the acclimation of photosynthesis, few of the
published experiments provide a large enough data-base to
be able to evaluate their contribution. There is evidence in
isolated cases indicating that selective regulation of
Rubisco or low rates of end-product synthesis contribute to
the acclimation of photosynthesis. Probably, by default,
many of the cases where acclimation has been observed
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
have included a contribution from earlier senescence
because the plants were not adequately fertilized. In future
experiments it will be important to pay careful attention to
the design of the experiments in order control the nitrogen
supply at a suitable level, to routinely include analyses not
only of carbohydrates but also of nitrogen metabolism
(Geiger et al. 1998b, 1999), and to monitor the expression
and protein levels of a much wider range of proteins
involved in photosynthesis (Nie et al. 1995a, 1995b;
Geiger et al. 1999) and housekeeping proteins, in order to
allow a clearer interpretation of the significance of
changes in single protein species. It will also be important
to address the possible contribution of diurnal rhythms in
transcripts in leaves (Cheng et al. 1998; Geiger et al.
1998b), to analyse the response at different stages through
the life-history of a single leaf (Miller et al. 1997) and of a
plant (Nie et al. 1995a, 1995b), and to combine the analy-
ses of events in primary metabolism with an analysis of
events related to leaf ageing and senescence. Transgenic
plants with altered expression of key steps underlying the
different explanations for acclimation including enzymic
steps in photosynthesis (see above) and processes leading
to senescence (Gan & Amasino 1997), and mutants
altered in sugar or nitrogen signalling could provide an
invaluable resource to dissect these complex interactions.
An unambiguous resolution of this problem will ulti-
mately depend on a better molecular understanding of
sugar signalling, nitrogen signalling and the regulation of
leaf ageing and senescence.
5.4 Photosynthate partitioning
Elevated [CO2] leads to increased levels of carbohydrates
in the leaf, with the major form being starch (Section
2.2). It has been tacitly assumed that the accumulation of
starch is a response to the accumulation of sugars.
Possible mechanisms could include a feed-back inhibi-
tion of sucrose synthesis or recycling of sucrose via
invertase (see above) leading to an accumulation of phos-
phorylated metabolites and stimulation of AGPase by an
increased 3PGA:phosphate ratio (for more details see
Stitt 1991), and increased expression of AgpS as a result
of sugar-mediated signalling (Müller-Röber et al. 1990;
Krapp & Stitt 1995; Nielsen et al. 1998).
Several studies, however, have found a marked increase
of starch in elevated [CO2] even though sugar levels do not
change significantly (Morin et al. 1992; Van Der Kooij &
De Kok 1996; Ludewig et al. 1998; Geiger et al. 1999) As
discussed in Section 3.7, AgpS expression is regulated by
signals from nitrate and phosphate metabolism. The
decrease of nitrate (see Sections 2.3 and 5.1) in elevated
[CO2] could be primarily responsible for the dramatic
increase of the AgpS transcript in elevated [CO2] (Geiger
et al. 1999), and contribute to the increase in starch accu-
mulation. This might also explain why the increase of
starch in elevated [CO2] is especially marked in nitrogen-
limited plants (see Section 2.2) and why sugars are present
in higher levels in well-fertilized plants than in nitrogen-

limited plants in ambient (Fichtner et al. 1993) and elevated
(Ferrario-Mery et al. 1997; Geiger et al. 1999) [CO2].
Any stimulation of starch accumulation and reduction of
sugar formation due to signals originating from nitrate or
other nutrients will affect the ability of the plant to initiate
changes in allocation that require sugar signalling. This
could result in some extremely complex interactions when
elevated [CO2] is provided to plants at a nutrient supply
where it results in large changes in the key pools of nutri-
ents and the metabolites formed from them. Careful analy-
ses of nitrogen metabolism in combination with changes
in allocation will be required to unravel these complex
interactions, and it will probably be useful to employ
genotypes with altered expression of selected genes
involved in nitrogen metabolism, and to use sophisticated
methods to monitor and control the supply of nutrients to
the plant, including the Ingestad method (Ingestad 1997)
in which the nutrient supply is increased in parallel with
the absolute rate of plant growth (see Pettersson et al.
1993; Pettersson & MacDonald 1994 for applications to
plants growing in elevated [CO2]).
5.5 Whole plant growth
The effect of elevated [CO2] on growth is strongly modu-
lated by the nitrogen supply (Section 2.3). In this final sec-
tion, we summarize some of the changes in photosynthesis,
carbon allocation and nitrogen allocation that underlie this
dramatic modulation of the response to elevated [CO2].
5.5.1 Growth rate
Elevated [CO2] leads to a sustained stimulation of photo-
synthesis, elevated levels of sugars, and supports increased
growth in well-fertilized plants, but not in nitrogen-defi-
cient plants (Section 2). Two interacting factors explain
why elevated [CO2] leads to a large increase of biomass in
well-fertilized tobacco, but has little or no effect on the
biomass of nitrogen-limited tobacco. First, as shown in
studies of tobacco transformants with decreased expres-
sion of Rubisco, alterations in the rate of photosynthesis
have a large effect on the rate of growth of well-fertilized
plants but have little or no effect on the rate of growth of
nitrogen-limited plants (Quick et al. 1993; Fichtner et al.
1993; Stitt & Schulze 1994). Second (Sections 2.1 and
5.3.1) elevated [CO2] produces a sustained stimulation of
photosynthesis and of sugar formation in well-fertilized
plants, but not in nitrogen-limited plants.
When nitrogen-replete plants are grown in elevated
[CO2], the total and individual amino acids and protein
remain high or even increase slightly, and the activities of
Rubisco and other Calvin cycle remain high or increase
slightly (Section 5.3.1). This allows an increased rate of
photosynthesis to be maintained, compared to plants grow-
ing in ambient [CO2]. The AgpS transcript remains rela-
tively low in elevated [CO2], and starch only increases
slightly compared to plants growing in ambient [CO2]
(Section 5.4). The increased rate of net photosynthesis and
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 609
the relatively low rates of starch accumulation result in
higher levels of sugars in the leaves, and make more photo-
synthate available for export to support growth. Although
it is possible that the sugars also act as signals to promote
growth (Section 4, Koch 1996; see also discussion in
Micallef et al. 1995), they do not act as an effective signal to
inhibit the expression of Rubisco or other proteins involved
in photosynthesis in these conditions (Section 5.3.1).
When plants with an intermediate or a limiting nitrogen
supply are grown in elevated [CO2], NR activity decreases
(Section 5.2.2), nitrate, amino acids and total protein
decrease (Section 5.1), the activities of Rubisco and other
Calvin cycle enzymes decrease (Section 5.3.1), and the
rate of photosynthesis in elevated [CO2] is only slightly
increased or even decreases compared to plants growing in
ambient [CO2] (Section 2.1). Expression of AgpS is
increased (Section 5.3.2), and larger amounts of carbon
accumulate as starch in elevated [CO2] (Section 2.2). The
additional starch that accumulates in elevated [CO2] in
nitrogen-limited plants will represent a substantial portion
of the net photosynthesis in freshly expanded leaves
(Fichtner et al. 1993). As a result, the amount of photosyn-
thate that is available for export to support growth will not
be significantly increased, and may even decrease. This,
together with possible effects of decreased transpiration on
the availability of nitrate in the soil (Section 2.6) may
explain why elevated [CO2] sometimes even leads to a
slight decrease of biomass in severely nitrogen-limited
plants (Section 2.3).
Ultimately the rate of growth will depend upon the num-
ber of active meristems, and rates of cell division and cell
elongation, and the duration of the division and elongation
phases at each individual meristem. Elevated [CO2] results
in a shortening of the cell cycle in the shoot and root meris-
tem (Kinsman et al. 1997). It will be important to deter-
mine to what extent this is solely due to an increased
supply of sugars to the meristem, and to what extent sugar-
or nutrient-mediated signalling modulates the activity at
the meristems. In this context, it might be noted that the
modulation of root meristem growth by internal and exter-
nal nitrate does not involve changes in the levels of amino
acids in the roots (see Section 3.9). It is more likely that the
carbon and nutritional status of the plant is sensed and
transduced via hormones or other signalling molecules.
5.5.2 Elevated carbon dioxide has an age-dependent effect
on the rate of plant growth
Any consideration of the effect of elevated [CO2] on the
physiology and growth of plants must, however, also
address time-dependent changes in the growth rate of
plants. In general, growth is rapid in young plants and then
decreases as the plant ages (see, e.g. Masle, Hudson &
Badger 1993; Geiger et al. 1998b). The effect of elevated
[CO2] on growth also depends on plant age. Studies with
alfalfa (Baysdorfer & Bassham 1985), five different wild
annual species (Garbutt et al. 1990), Poa alpina (Baxter
et al. 1994), Arabidopsis (Van de Kooij & de Kok 1996)
610 M. Stitt and A. Krapp
and tobacco (Geiger et al. 1998b) have shown that long-
term exposure to elevated [CO2] leads to a stimulation of
the current relative growth rate in young plants, whereas
the relative growth rate in older plants is unaffected.
Several studies of trees have also reported that the increase
in biomass is largely due to increased rates of growth in the
first year of exposure to elevated [CO2], and that growth is
stimulated less or is not increased at all in the subsequent
years (Cuelemans, Jiang & Shao 1994; Lee & Jarvis 1995;
Tissue et al. 1997).
Young seedlings are typically ‘source’-limited, and con-
tain far lower levels of sugars than older plants (Geiger
et al. 1998b). This provides one simple explanation why
elevated [CO2] leads to a larger stimulation of metabolism
and growth in seedlings than in older plants. The fall off of
the growth rate in elevated [CO2] in older plants may often
be partly due to an earlier exhaustion of the nutrient supply.
This problem can, of course, be counteracted by supplying
higher amounts of nutrient, for example by growing the
plants in hydroponic culture with controlled nitrogen lev-
els, or by increasing the nutrient supply in a parallel with
the plant size (see above). However, the gradual fall-off in
the growth rate of plants is probably also partly due to
endogenous ontogenetic factors. When tobacco plants
were grown in hydroponic culture at an excess nitrogen
supply, the growth stimulation by elevated [CO2] was
observed for a longer time than in sand or soil culture, but
the stimulation nevertheless still disappeared in older
plants (M. Geiger, P. Walsch-Liu, C. Engels and M. Stitt,
unpublished). Since elevated [CO2] allows plants, espe-
cially those with determinate life histories, to move
through their life history at a faster rate it will lead to an
earlier onset of an ontogenetic decline in growth rates, to
which several factors may be contributing. A a plant
becomes larger, an increasing part of the biomass is com-
mitted to stems, older roots and other support structures,
and a decreasing part to leaves and young roots which are
responsible for the acquisition and assimilation of carbon
dioxide and nutrients. Self-shading of leaves, or physical
restriction of root growth (Arp 1991), can lead to a further
decrease in growth rates. Especially in determinate plants,
the number of growth points at active meristems may not
keep pace with the production of assimilatory structures.
Seedlings grow rapidly and exponentially, and over a
period of days a relatively small 20% increase in the rela-
tive growth rate can result in a doubling of the plant
biomass (Geiger et al. 1998b). The marked increase of
biomass of older plants in elevated [CO2] therefore reflects
a small increase in the rate of growth at an earlier period of
their life history. This age-dependent effect of elevated
[CO2] on the rate of growth has enormous implications for
the design and interpretation of experiments that investigate
the effect of elevated [CO2] on metabolic and physiological
processes (see also Körner 1995 for a discussion of their
impact on up-scaling to ecosystem responses). Firstly, even
though plants show striking differences in their biomass,
the underlying changes in the rate of growth and hence in
molecular and physiological processes may be quite small,
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
and difficult to detect. Secondly, in order to interpret the
significance of changes in physiological and molecular
parameters in elevated [CO2], it is essential to relate them to
the current growth rate of the plant. Many comparisons of
photosynthesis, allocation and nitrogen metabolism in
older plants in ambient and elevated [CO2] may have been
studying two sets of plants which have a similar current
growth rate but have exhausted the available nutrient sup-
ply to a varying extent and/or are at a different stage in
their life history. This, for example, may be another reason
for the apparent anomaly that plants in elevated [CO2]
have lower NR activity and lower levels of nitrogen, amino
acids and protein (see Section 2.4) but are apparently
growing faster than plants in ambient [CO2].
There are important endogenous changes in regulation
during development, that could be modified by elevated
[CO2], leading to further complex interactions. For example,
shoot morphogenesis is typically characterized by three
developmental phases, juvenile, adult and reproductive
(Poethig 1990). Tsai et al. (1997) used antisense RbcS
tobacco transformants as a experimental system to decrease
the rate of photosynthesis and the levels of sugars, and
observed that this resulted in the production of a larger num-
ber of leaves in the juvenile phase. This result implies that
signals related to the levels of sugars may modulate the tran-
sition from the juvenile to the adult phase (Tsai et al. 1997).
It will be important to investigate whether this shift in
ontogeny occurs earlier in elevated [CO2], and discover
whether it affects the subsequent ability to maintain
increased rates of photosynthesis and growth in elevated
[CO2]. It will also be important to investigate whether this
developmental shift is solely due to changes in the levels of
carbohydrates, or if changes in other nutrients including
nitrogen metabolites are also involved. For these reasons, it
may be misleading to apply an oversimplified schemata
when considering the contribution of ontogenetic drift to the
response of tissue composition of growth to elevated [CO2].
5.5.3 Changes in the allocation of biomass in elevated
carbon dioxide may sometimes be mediated by changes in
nutrient status
Elevated [CO2] leads to changes in biomass allocation and
phenology which can include an increase of the root:shoot
ratio, tillering, increased branching, and earlier flowering
(Bazzaz & Fajer 1992; Tissue et al. 1997).
Some of these changes may be mediated via changes in
the nutrient status. For example, signals from nitrate regu-
late root:shoot allocation in tobacco and Arabidopsis
(Section 3.9). Elevated [CO2] typically leads to a decrease
of nitrate (Section 2.4) especially if the nitrogen supply is
not super-optimal (Section 5.1), and this or other nitrogen-
mediated signals could be responsible for the change in
shoot : root allocation often reported in elevated [CO2].
When N. plumbaginifolia was grown hydroponically with
a high nitrate supply, elevated [CO2] did not affect the
root : shoot ratio (Ferrario-Mery et al. 1997). Elevated
[CO2] led to a marked increase of the root : shoot ratio in

nitrogen-limited Poa alpina, whereas the ratio decreased in
well-fertilized plants (Baxter et al. 1997). Elevated [CO2]
also leads to an increase of the root : shoot ratio in nitro-
gen-limited tobacco, but not in well-fertilized tobacco
(Geiger et al. 1999). When the rate of photosynthesis was
altered by inhibiting expression of Rubisco, root : shoot
allocation was only altered compared to wild-type plants
when the plants were grown in high nitrate (Fichtner et al.
1993), when the lower rate of photosynthesis in the trans-
formants led to a marked increase of nitrate in the plants
(Stitt & Schulze 1994). The increase of tillering (Baxter
et al. 1997), branching (Tissue et al. 1997), tuber initiation
(see Heineke and Sonnewald, 1999) and earlier flowering
(Bazzaz & Fajer 1992) in elevated [CO2] may also not be
exclusively due to the increased carbohydrate levels, but
involve an interaction with nitrogen.
In conclusion, it is essential to investigate the changes in
nitrogen metabolism in parallel with investigations of photo-
synthesis, carbon allocation, and growth in elevated [CO2].
Unless the effect of elevated [CO2] on the plant nutrient
status is known, there is a serious risk that the changes in
other parameters will be misinterpreted. A detailed analysis
of nitrogen metabolism is needed in order to allow the con-
tributions of sugar-mediated signalling, nitrogen-induced
senescence, and ontogenetic drift to be disentangled, and it
is likely that some of the responses observed under elevated
[CO2] will be due to more specific changes initiated as a
result of signals emanating from the changes in nitrate levels
and in nitrogen metabolism in the plant.
ACKNOWLEDGMENTS
The authors own research on elevated carbon dioxide has
been supported by the Deutsche Forschungsgemeinschaft.
We are grateful to G. Corruzzi and H. Lam, U. Sonnewald.
W. Frommer, C. Engels and D. Heineke, J. Smeekens, T.
Miller and B. Forde for making results accessible to us
before publication.
REFERENCES
Andrews M. (1986) The partitioning of nitrate assimilation
between root and shoot of higher plants. Plant, Cell and
Environment 9, 511–519.
Andrews M., Morton J.D., Liefering M. & Bisset L. (1992) The
partitioning of nitrate assimilation between root and shoot of a
range of temperate cereals and pasture grasses. Annals of Botany
70, 271–276.
Appeldoorn N.J.G., de Bruijn S.M., Koot-Gronsfeld E.A.M.,
Visser R., Vreugdenhil D. & van de Plas L.H.W. (1997)
Developmental changes of enzymes involved in conversion of
sucrose to hexose phosphates during early tuberisation ot
potato. Planta 202, 220–226.
Arp W.-J. (1991) Effects of source-sink relations on photosynthetic
acclimation to elevated carbon dioxide. Plant, Cell and
Environment 14, 869–876.
Arp W.J., Mierlo J.E.M., Berendse F. & Snijders W. (1998)
Interactions between elevated carbon dioxide concentration,
nitrogen and water: effects on growth and water use of six peren-
nial plant species. Plant, Cell and Environment 21, 1–11.
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 611
Aslam M., Travis R.L. & Huffacker R.C. (1992) Comparative
kinetics and reciprocal inhibition of nitrate and nitrite uptake in
roots of uninduced and induced barley seedlings. Plant
Physiology 99, 1124–1133.
Aslam M., Travis R.L. & Rains D.W. (1996) Evidence for substrate
induction of a nitrate efflux system in barley. Plant Physiology
112, 1167–1175.
Atkinson C.J., Taylor J.M., Wilkins D. & Besford R.T. (1997)
Effects of elevated carbon dioxide on chloroplast components,
gas exchange and growth of oak and cherry. Tree Physiology
17, 319–325.
Bachmann M., Huber J.L., Liao P.-C., Gage D.A. & Huber S.C.
(1996) The inhibitor protein of phosphorylated nitrate reductase
from spinach (Spinacia oleracea) leaves is a 14-3-3 protein.
FEBS Letters 387, 127–131.
Bassirirad H., Reynolds J.F., Virginia R.A. & Brumelle M.H.
(1997) Growth and root nitrate and phosphate uptake capacity
of three desert species in response to atmospheric carbon
dioxide enrichment. Australian Journal of Plant Physiology
24, 353–358.
Bassirirad H., Thomas R.B., Reynolds J.F. & Strain B.R. (1996)
Differential responses of root uptake kinetics of ammonium
and nitrate to enriched atmospheric carbon dioxide concentra-
tion in field grown loblolly pine. Plant, Cell and Environment
19, 367–371.
Baxter R., Ashenden T.W. & Farrar J. (1997) Effect of elevated
carbon dioxide and nutrient status on growth, dry matter parti-
tioning and nutrient content of Poa alpina var. vivpara L.
Journal of Experimental Botany 48, 1477–1486.
Baxter R., Grantley M., Ashenden T.W. & Farrar J. (1994) Effects
of elevated carbon dioxide on three grass species from montane
pasture. II Nutrient allocation and efficiency of nutrient use.
Journal of Experimental Botany 45, 1267–1278.
Baysdorfer C. & Bassham J.A. (1985) Photosynthetic supply and
utilisation in alfalfa: a developmental shift from a source to a
sink limitation of photosynthesis. Plant Physiology 77, 313–317.
Bazzaz F.A. & Fajer E.D. (1992) Plant life in a CO2-rich world.
Scientific American 266, 68–74.
Bernier G., Havelange A., Houssa C., Petitjean A. & Lejeune P.
(1993) Physiological signals that induce flowering. Plant Cell
5, 1147–1155.
Besford R.T. & Hand D.W. (1989) The effects of carbon dioxide
enrichment and nitrogen oxides on some Calvin cycle enzymes
and nitrate reductase in glasshouse lettuce. Journal of
Experimental Botany 40, 329–336.
Besford R.T., Ludwig I.J. & Withers A.C. (1990) The greenhouse
effect: acclimation of tomato plants growing in high carbon diox-
ide. Photosynthesis and ribulose-1,5-bisphosphate carboxylase
protein. Journal of Experimental Botany 41, 925–931.
Bloom A.J., Chapin F.S. & Mooney H.A. (1985) Resource limita-
tion in plants – an economic analogy. Annual Review of
Ecological Systems 16, 363–392.
Bloom A.J., Sukrapanna S.S. & Warner R.L. (1992) Root respira-
tion associated with ammonium and nitrate absorption and
assimilation by barley. Plant Physiology 99, 1294–1301.
Borisjuk L., Walenta S., Weber H., Müller-Klieser W. & Wobus U.
(1998) High resolution histological mapping of glucose concen-
trations in the developing cotyledons of Vicia faba in relation to
mitotic activity and storage processes: glucose as a possible
developmental trigger. Plant Journal 15, 583–591.
Botrel A. & Kaiser W. (1997) Nitrate reductase activation status in
barley roots in relation to the energy and carbohydrate status.
Planta 201, 496–501.
Bowes G. (1996) Elevated carbon dioxide. In Advances in
Photosynthesis, Vol. 3. Environmental Stress and Photosynthesis
(ed. N. Baker), pp. 387–407. Academic Press, Dordrecht.
Bowler J.M. & Press M.C. (1996) Effects of elevated carbon
612 M. Stitt and A. Krapp
dioxide, nitrogen form and concentration on growth and
photosynthesis of a fast- and slow-growing grass. New
Phytologist 132, 391–401.
Brooks A. (1986) Effects of phosphorus nutrition on ribulose-1,5-
bisphosphate carboxylase activation, photosynthetic quantum
yield and amounts of Calvin cycle enzymes in spinach leaves.
Australian Journal of Plant Physiology 13, 221–237.
Brouquisse R., James F., Pradet A. & Raymond P. (1992)
Asparagine metabolism and nitrogen distribution during protein
degradation in sugar-starved maize roots. Planta 188, 384–395.
Brouwer R. (1962) Nutrient influences on the distribution of the dry
matter in the plant. Netherlands Journal of Agricultural Sciences
10, 399–408.
Cavicchioli R., Chiang R.C., Kalman L.V. & Gunsalus R.P.
(1996) Role of the periplasmic domain of Escherichia coli
NarX sensor-transmitter protein in nitrate-dependent signal
transduction and gene regulation. Molecular Microbiology 21,
901–911.
Cavicchioli R., Schoeder I., Consanti M. & Gunsalus R.P. (1995)
The NarX and NarQ sensor-transmittor proteins of Escherichia
coli each require two conserved histidines for nitrate-dependent
signal transduction to NarL. Journal of Bacteriology 177,
2416–2424.
Champigny M.L., Brauer M., Bismuth E., ThiManh C., Siegl G.,
Van Quy L. & Stitt M. (1992) The short term effect of NO3– and
NH4+ assimilation on sucrose synthesis in leaves. Journal of
Plant Physiology 139, 361–368.
Cheng C.L., Acedo G.N., Cristinsin M. & Conkling M.A. (1992)
Sucrose mimics the light induction of Arabidopsis nitrate reduc-
tase gene transcription. Proceedings of the National Academy of
Science of the USA 89, 1861–1864.
Cheng S.-H., Moore D. & Seeman J.R. (1998) Effects of short- and
long-term elevated carbon dioxide on the expression of ribilose-
1,5-bisphosphate carboxylase/oxygenase genes and carbohy-
drate accumulation in leaves of Arabidopsis thaliana (L.) Heynh.
Plant Physiology 116, 715–723.
Chevalier C., Boureois E., Just D. & Raymond P. (1996) Metabolic
regulation of asparagine synthetase gene expression in maize
root tips. Plant Journal 9, 1–11.
Chiang R.C., Cavicchioli R. & Gunsalis R.P. (1997) Locked-on and
locked-off signal transduction mutations in the periplasmic
domain of the Escherichia coli NARQ and NARX sensors affect
nitrate and nitrate dependent regulation by NARL and NARP.
Molecular Microbiology 24, 1049–1060.
Chiou T.-J. & Bush D.R. (1998) Sucrose is a signal molecule in
assimilate partitioning. Proceedings of the National Academy of
Sciences USA 95, 4784–4788.
Chollet R., Vidal J. & O’Leary M.H. (1996) Phosphoenolpyruvate
carboxylase: a ubiquitous, highly regulated enzyme in plants.
Annual Review of Plant Physiology and Plant Molecular Biology
47, 273–298.
Chu C.C., Coleman J.S. & Mooney H.A. (1992) Control of biomass
partitioning between roots and shoot: atmospheric carbon diox-
ide enrichment and the acquisition and allocation of carbon and
nitrogen in wild radish. Oecologia 89, 580–587.
Chu C.C., Field C.B. & Mooney H.A. (1996) Effects of carbon
dioxide and nutrient enrichment on tissue quality of two
Californian annuals. Oecologia 107, 433–440.
Clarkson D.T. & Lüttge U. (1991) Mineral nutrition: inducible and
repressible transport systems. Progress in Botany 52, 61–63.
Coleman J.S. & Bazzaz F.A. (1992) Effects of CO2 and temperature
on growth and resource use of co-occurring C3 and C4 annuals.
Ecology 73, 1244–1259.
Coleman J.S., McConnaughay K.D.M. & Bazzaz F.A. (1993)
Elevated carbon dioxide and plant nitrogen use. Is reduced tissue
nitrogen concentration size dependent. Oecologia 93, 195–200.
Coleman J.S., Rochefort L., Bazzaz F.A. & Woodward F.I. (1991)
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Atmospheric carbon dioxide, plant nitrogen status and the sus-
ceptibility of plants to an acute increase in temperature. Plant,
Cell and Environment 14, 667–674.
Conroy J. & Hocking P. (1993) Nitrogen nutrition of C3 plants at ele-
vated atmospheric CO2 concentrations. Physiologia Plantarum 89,
570–576.
Conroy J.P., Milham P.J. & Barlow E.W.R. (1992) Effect of nitrogen
and phosphorus availability on the growth response of Eucalyptus
grandis to high CO2. Plant, Cell and Environment 15, 843–847.
Cotrufo M.F., Ineson P.- & Scott A. (1998) Elevated carbon dioxide
reduces the nitrogen concentration of plant tissues. Global
Change Biology 4, 43–54.
Crawford N.M. (1995) Nitrate: nutrient and signal for plant growth.
Plant Cell 7, 859–868.
Crawford N.M. & Arst H.N. Jr (1993) The molecular genetics of
nitrate assimilation in fungi and plants. Annual Review of
Genetic 27, 115–146.
Crete P., Caboche M. & Meyer C. (1997) Nitrite reductase expres-
sion is regulated at the post-transcriptional level by the nitrogen
source in Nicotiana plumbaginifolia and Arabidopsis thaliana.
Plant Journal 11, 625–634.
Cuelemans R., Jiang X.N. & Shao B.Y. (1994) Growth and physiol-
ogy of one year old poplar under elevated atmospheric carbon
dioxide levels. Annals of Botany 75, 609–917.
Curtis P.S., Drake B.G. & Whigham D.F. (1989) Nitrogen and car-
bon dynamics in C3 and C4 estuarine marsh plants grown under
elevated CO2 in situ. Oecologia 78, 297–301.
Dancer J.E., Hatzfeld W.-D. & Stitt M. (1990) Cytosolic cycles reg-
ulate the turnover of sucrose in heterotrophic cell suspension cul-
tures of Chenopodium rubrum L. Planta 182, 223–231.
Daniel-Vedele F. & Caboche M. (1993) A tobacco cDNA clone
encoding a GATA-1 zinc finger protein homologous to regulators
of nitrogen metabolism in fungi. Molecular and General
Genetics 240, 365–373.
Daniel-Vedele F. & Caboche M. (1996) Molecular analysis of nitrae
assimilation in higher plants. Comptes Rendus de l’Academie
Des Sciences, Series III 319, 961–968.
Daniel-Vedele F., Filleur S. & Caboche M. (1998) Nitrate transport:
a key step in nitrate assimilation. Current Opinion in Plant
Biology 1, 235–239.
Darwin A., J. & Stewart V. (1995) Nitrate and nitrite regulation of
the Fnr, dependent aeg-46.5 promoter of Escherichia coli K-12 is
mediated by competition between homologous response regula-
tors (NarL and NarP) for a common DNA binding site. Journal of
Molecular Biology 251, 15–29.
Delgrado E., Mitchell R.A.C., Parry M.A.J., Driscoll S.P., Mitchell
V.J. & Lawlor D.W. (1994) Interacting effects of CO2 concentra-
tion, temperature and nitrogen supply to leaves on photosynthe-
sis and composition of winter wheat leaves. Plant, Cell and
Environment 17, 1205–1213.
Delhon P., Gojon A., Tillard P. & Passama L. (1995) Diurnal regula-
tion of NO3 uptake in soybean plants. Journal of Experimental
Botany 46, 1585–1594.
Den-Hertog J., Stulen I., Fonseca E. & Delea P. (1996) Modulation of
carbon and nitrogen allocation in Urtica dioica and Plantago major
by elevated CO2: Impact of accumulation of nonstructural carbohy-
drates and ontogenetic drift. Physiologia Plantarum 98, 77–88.
Dever T.E., Feng L., Wek R.C., Cigan A.M., Donahue T.F. &
Hinnebusch A.G. (1992) Phosphorylation of initiation factor 2-
alpha by protein kinase GCN2 mediates gene-specific transla-
tional control of GCN4 in yeast. Cell 68, 585–596.
Di Laurenzio L., Wysocka-Diller J., Malamy J.E., Pysh L., Helariutta
Y., Freshour G., Hahn M.G., Feldmann K. & Benfey P. (1996) The
SCRAREROW gene regulates an asymetric cell division that is
essential for generating the radical organization of the Arabidopsis
root. Cell 86, 423–433.
Diaz A., Lacuestra M. & Munoz-Rueda A. (1996) Comparative

effects of phosphinothricin on nitrate and ammonium assimila-
tion and on anapleurotic carbon dioxide fixation in N-deprived
barley plants. Journal of Plant Physiology 149, 9–13.
Dietz K.-J., Hollenbach B. & Hellwege E. (1994) The epidermis of
barley is a dynamic intermediary storage compartment of carbo-
hydrates, amino acids and nitrate. Physiologia Plantarum 92,
31–36.
Dijkwel P.P., Kock P.A.M., Bezemer R., Weisbeek P.J. & Smeekens
S.C.M. (1997) Sucrose control of phytochrome A signaling in
Arabidopsis. Plant Cell 9, 583–595.
Drake B.G., Gonzàlez-Meler M.A. & Long S.P. (1997) More effi-
cient plants: a consequence of elevated carbon dioxide? Annual
Review of Plant Physiology and Plant Molecular Biology 48,
607–640.
Drew M.C. & Saker L.R. (1975) 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.
Journal of Experimental Botany 26, 79–90.
Dubois F., Brugière N., Sangwan R.S. & Hirel B. (1996)
Localization of tobacco cytosolic glutamine synthetase enzymes
and the corresponding transcripts show organ- and cell-specific
patterns of protein synthesis and gene expression. Plant
Molecular Biology 31, 803–817.
Duff S.M.G. & Chollet R. (1995) In vivo regulation of wheat-leaf
phosphoenolpyruvate carboxylase by reversible phosphoryla-
tion. Plant Physiology 107, 775–782.
Eamus D. & Jarvis P.G. (1989) The direct effects of increase in the
global atmospheric CO2 concentration on natural and commer-
cial temperate trees and forests. Advances in Ecological
Research 19, 1–55.
El-Kohen A. & Mousseau M. (1994) Interactive effects of elevated
carbon dioxide and mineral nutrition on growth and carbon diox-
ide exchange of sweet chestnut seedlings. Tree Physiology 14,
679–690.
Evans J.R. (1989) Photosynthesis and nitrogen relationships in
leaves of C3-plants. Oecologia 78, 9–19.
Evans J.R. (1996) Influence of the environment on the composition
of the photosynthetic apparatus. In Advances in Photosynthesis,
Vol. 3. Environmental Stress and Photosynthesis (ed. N. Baker),
pp. 281–304. Academic Press, Dordrecht.
Fangmeier A., Grueters U., Hoegy Vermehren B. & Jaeger H.J.
(1997) Effects of elevated carbon dioxide, nitrogen suply and
tropospheric ozone on spring wheat. II Nutrients. Environmental
Pollution 96, 43–59.
Faria T., Wilkins D., Besford R.T., Vaz M., Pereira J.S. & Chaves
M.M. (1996) Growth at elevated carbon dioxide leads to down
regulation of photosynthesis and altered response to high temper-
ature in Quercus suber seedlings. Journal of Experimental
Botany 47, 1755–1761.
Faure J.D., Jullien M. & Caboche M. (1994) Zea3: a pleiotropic
mutation affecting cotyledon development, cytokinin resis-
tance and carbon-nitrogen metabolism. Plant Journal 5,
481–491.
Ferrario S., Valadier M.H. & Foyer C.H. (1996) Short term mod-
ulation of nitrate reductase activity by exogenous nitrate in
Nicotiana plumbaginifolia and Zea mays. Planta 199,
366–371.
Ferrario S., Valadier M.-H., Morot-Gaudry J.-F. & Foyer C.H.
(1995) Effects of constitutive expression of nitrate reductase in
transgenic Nicotiana plumbaginifolia L. in response to varying
nitrogen supply. Planta 196, 288–294.
Ferrario-Mery S., Thibaud M.-C., Betsche T., Valadier M.-H. &
Foyer C.H. (1997) Modulation of carbon and nitrogen
metabolism, and of nitrate reductase, in untransformed Nicotiana
plumbaginifolia during CO2 enrichment of plants grown in pots
and hydroponic culture. Planta 202, 510–521.
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 613
Fetene M. & Beck E. (1993) Reversal of sink-source relations in
Urtica dioca L. plants by increasing cytokinin import into the
shooot. Botanical Acta 106, 235–240.
Fichtner K. & Schulze E.-D. (1992) The effect of nitrogen nutrition
on annuals originating from habitats of different nitrogen avail-
ability. Oecologia 92, 236–241.
Fichtner K., Quick W.P., Schulze E.D., Mooney H.A., Rodermel
S.R., Bogorad L. & Stitt M. (1993) Decreased ribulose-1,5-bis-
phosphate carboxylase-oxygenase in transgenic tobacco trans-
formed with ‘antisense’ rbcS. V. Relationship between
photosynthetic rate, storage strategy, biomass allocation and veg-
etative plant growth at three different nitrogen supplies. Planta
190, 1–9.
Fieuw S., Müller-Röber B., Gálvez S. & Willmitzer L. (1995)
Cloning and expression analysis of the cytosolic NADP+-depen-
dent isocitrate dehydrogenase from potato. Plant Physiology
107, 905–913.
Fischer W.N., Andre B., Rentsch D., Krolkiewicz S., Tegeder M.,
Breitkreuz K. & Frommer W.B. (1998) Amino acid transport in
plants. Trends in Plant Science 3, 188–195.
Fischer B.U., Frehner M., Hebeisen T., Zanetti S., Stadelmann F.,
Luscher A., Hartwig U.A., Hendrey G.R., Blum H. & Nosberger
J. (1997) Source-sink relations in Lolium perenne L. as reflected
by carbohydrate concentrations in leaves and pseudostems dur-
ing regrowth in a free air carbon dioxide enrichment experiment.
Plant, Cell and Environment 20, 945–952.
Fonseca F., Bowsher C. & Stulen I. (1997) Impact of elevated atmo-
spheric carbon dioxide on nitrate reductase transcription and
activity in leaves and roots of Plantago major. Physiologia
Plantarum 100, 940–948.
Forde B.G. & Clarkson D.T. (1999) Nitrate and ammonium nutri-
tion of plants: physiological and molecular perspectives.
Advances in Botanical Research in press.
Foyer C.H., Lescure J.C., Lefèbvre C., Morot-Gaudry J.F. &
Vaucheret H. (1994a) Adaptations of photosynthetic electron
transport, carbon assimilation and carbon partitioning in trans-
genic Nicotiana plumbaginifolia plants to changes in nitrate
reductase activity. Plant Physiology 104, 171–178.
Foyer C.H., Noctor G., Lelandai M., Lescure J.C., Valadier M.H.,
Boutin J.P. & Horton P. (1994b) Short-term effects of nitrate,
nitrite and ammonium assimilation on photosynthesis, carbon
partitioning and protein phosphorylation in maize. Plant
Physiology 192, 211–220.
Franco A.R., Cardenas J. & Fernandez E. (1987) Involvement of
reversible inactivation of nitrate reductase in the regulation of
enzyme levels in Chlamydomonas reinhardtii. Plant Physiology
84, 665–669.
Fredeen A.L., Raab T.K., Rao I.M. & Terry N. (1990) Effects of
phosphorus nutrition on photosynthesis in Glycine max. Planta
181, 399–405.
Galangau F., Daniel-Vedele F., Moureaux T., Dorbe M.F.,
Leydecker M.T. & Caboche M. (1988) Expression of leaf
nitrate reductase gene from tomato and tobacco in relation to
light dark regimes and nitrate supply. Plant Physiology 88,
383–388.
Galtier N., Foyer C.H., Murchie E., Alred R., Quick P., Voelker
T.A., Thepenier C., Lasceve G. & Betsche T. (1995) Effects of
light and atmospheric carbon dioxide enrichment on photosyn-
thesis and carbon partitioning in the leaves of tomato
(Lycopersicon esculentum L.) plants overexpressing sucrose
phosphate synthase. Journal of Experimental Botany 46,
1335–1344.
Galvan A., Quesada A. & Fernandez E. (1996) Nitrate and nitrite
are transported by different specific transport systems and a bis-
pecific transporter in Chlamydomonas reinhardtii. Journal of
Biological Chemistry 271, 2088–2092.
Gan S., Amasino R.M. (1997) Making sense of senescence.
614 M. Stitt and A. Krapp
Molecular genetic regulation and manipulation of leaf senes-
cence. Plant Physiology 113, 313–319.
Garbutt K., Williams W.E. & Bazzaz F.A. (1990) Analysis of the
differential response of five annuals to elevated carbon dioxide
during growth. Ecology 7, 1185–1194.
Gebauer G., Melzer A. & Rehder H. (1984) Nitrate content and
nitrate reductase activity in Rumex obtusifolia L. I. Differences in
organs and diurnal changes. Oecologie 63, 136–142.
Gebauer R.L.E., Reynolds J., F., Strain B. R. (1996) Allometric
relations and growth in Pinus taeda: the effect of elevated CO2
and changing N availability. New Phytologist 134, 85–93.
Geigenberger P. & Stitt M. (1993) Sucrose synthase catalyses a
readily reversible reaction in vivo in developing potato tuber and
other plant tissues. Planta 189, 329–339.
Geigenberger P., Lerchl J., Stitt. M. & Sonnewald U. (1996)
Phloem-specific expression of pyrophosphatase inhibits long-
distance transport of carbohydrates and amino acids in tobacco
plants. Plant, Cell and Environment 19, 43–55.
Geiger M., Haake V., Ludewig F., Sonnewald U. & Stitt M. (1999)
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
and Environment in press.
Geiger M., Stitt M. & Geigenberger P. (1998a) Metabolism in slices
from growing tubers reacts differently to addition of sucrose and
glucose. Planta 206, 234–244.
Geiger M., Walch-Piu L., Harnecker J., Schulze E.-D., Ludewig F.,
Sonnewald U., Scheible W.-R. & Stitt M. (1998b) Enhanced car-
bon dioxide leads to a modified diurnal rhythm of nitrate reduc-
tase activity in older plants, and a large stimulation of nitrate
reductase activity and higher levels of amino acids in higher
plants. Plant, Cell and Environment 21, 253–268.
George V., Gerant D. & Dizengremel P. (1996) Photosynthesis,
Rubisco activity and mtichondrial malate oxidation in peducu-
late oak seedlings grown under present and elevated atmospheric
carbon dioxide concentrations. Annales Des Sciences
Forestrieres (Paris) 53, 469–474.
von Ghannoum O., Caemmerer S., Barlow E.W.R. & Conroy J.P.
(1997) The effect of carbon dioxide enrichment on the growth,
morphology and gas exchanges of a C-3 (Panicum laxum) and a
C-4 (Panicum antidotale) grass. Australian Journal of Plant
Physiology 24, 227–237.
Godt D.E., Riegel A. & Roitsch T. (1995) Regulation of sucrose
synthase expression in Chenopodium rubrum: Characterization
of sugar induced expression in photoautotrophic suspension cul-
tures and sink tissue specific expression in plants. Journal of
Plant Physiology 146, 231–238.
Gojon A., Dapoigny L., Lejay L., Tillard P. & Rufty T.W. (1998)
Effects of genetic modification of nitrate reductase expression on
15
NO3- uptake and reduction in Nicotiana plants. Plant, Cell and
Environment 21, 43–53.
Goldman B.S., Lin J.T. & Stewart V. (1994) Identification and
structure of the nasR gene encoding a nitrate- and nitrite-respon-
sive positive regulator of nasFEDCBA (nitrate assimilation)
operon expression in Klebsiella pneumoniae M5a1. Journal of
Bacteriology 176, 5077–5085.
Goldschmidt E.E. & Huber S.C. (1992) Regulation of photosynthe-
sis by end-product accumulation in leaves of plants storing
starch, sucrose and hexose sugars. Plant Physiology 99,
1443–1448.
Graham I.A. (1996) Carbohydrate control of gene expression in
higher plants. Research in Microbiology 147, 572–580.
Granato T.C. & Raper C.D. Jr (1989) Proliferation of maize roots in
response to localised supply of nitrate. Journal of Experimental
Botany 40, 263–275.
Gries C., Kimball B.A. & Idso S.B. (1993) Nutrient uptake during
the course of a year by sour orange trees growing in ambient and
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
elevated carbon dioxide concentrations. Journal of Plant
Nutrition 16, 129–147.
Grime J.P., Campbell B.D., Mackey J.M.L. & Crick J.C. (1991)
Root plasticity, nitrogen capture and competitive ability. In Plant
Root Growth: an Ecological Perspective (ed. D. Atkinson), pp.
381–397. Blackwell Scientific Publications, Oxford, UK.
Gunderson C.A., Norby R.J. & Wullschleger S.D. (1993) Foliar gas
exchange of two deciduous hardwoods during three years of
growth in elevated CO2: no loss of photosynthetic enhancement.
Plant, Cell and Environment 16, 797–807.
Habash D.Z., Paul M.J., Parry M.A.J., Keys A.J. & Lawlor D.W.
(1995) Increased capacity for photosynthesis in wheat grown at
elevated carbon dioxide: the relationship between electron trans-
port and carbon metabolism. Planta 197, 482–489.
Hartwell J., Smith L.H., Wilkins M.B., Jenkins G.I. & Nimmo H.G.
(1996) Higher plants phosphoenolpyruvate carboxylase kinase is
regulated at the level of translatable mRNA in response to blue
light or a circadian rhythm. Plant Journal 10, 1071–1078.
Heim U., Weber H., Bäumlein H. & Wobus U. (1993) A sucrose-
synthase gene of Vicia faba L. expression pattern in relation to
starch synthesis and metabolic regulation. Planta 191, 394–401.
Heineke D., Kauder F., Frommer W., Kühn C., Gillissen B.,
Ludewig F. & Sonnewald D. (1999) Application of transgenic
plants in understanding responses to atmospheric change. Plant,
Cell and Environment Special Edition 22, 623–628.
Heldt H.W. (1996) Pflanzenbiochemie, pp. 285–299. Spektrum
Akademische-Verlag, Heidelberg.
Herbers K., Meuwly P., Frommer W.B., Metraux J.-P. & Sonnewald
U. (1996) Systemic acquired resistance mediated by the ectopic
expression of invertase: possible hexose sensing in the secretory
pathway. Plant Cell 8, 793–803.
Hilhorst H.W.M. & Karssen C.M. (1989) Nitrate reductase inde-
pendent stimulation of seed germination in Sisymbrium offici-
nale L. (hedge mustard) by light and nitrate. Annals of Botany
(Lond.) 63, 131–137.
Hirner B., Fischer W.N., Rentsch D., Kwart M. & Frommer W.B.
(1998) Developmental control of proton/amino acid permease
gene expression during seed development of Arabidopsis. Plant
Journal 14, 535–544.
Hocking P.J. & Meyer C.P. (1985) Responses of Noogoora burr
(Xanthium occidentale Bertol.) to nitrogen supply and carbon
dioxide enrichment. Annals of Botany (n.S.) 55, 835–844.
Hocking P.J. & Meyer C.P. (1991a) Effects of CO2 enrichment and
nitrogen stress on growth and partitioning of dry matter and
nitrogen in wheat and maize. Australian Journal of Plant
Physiology 18, 339–356.
Hocking P.J. & Meyer C.P. (1991b) Carbon dioxide enrichment
decreases critical nitrate and nitrogen concentrations in wheat.
Journal of Plant Nutrition 14, 571–584.
Hoff T., Truong H.-N. & Caboche M. (1994) The use of mutants
and transgenic plants to study nitrate metabolism. Plant, Cell and
Environment 17, 486–506.
Hofstra R., Lanting L. & De Visser R. (1985) Metabolism of Urtica
dioica as dependent on the supply of mineral nutrients.
Physiologica Plantarum 63, 13–18.
Holaday A.S., Martindale W., Alred R., Brooks A. & Leegood R.C.
(1992) Changes in activities of enzymes in carbon metabolism in
leaves during exposure to low temperature. Plant Physiology 75,
561–565.
Huang N.C., Chiang C.S., Crawford N.M. & Tsay Y.I. (1996) Chl1
encodes a component of the low affinity nitrate uptake system in
Arabidopsis and shows cell specific expression in roots. Plant
Cell 8, 2183–2191.
Huber S.C. & Huber J.L. (1996) Role and regulation of sucrose-
phosphate-synthase in higher plants. Annual Review of Plant
Physiology and Plant Molecular Biology 47, 431–444.
Huppe H.C. & Turpin D.H. (1994) Integration of carbon and nitro-

gen metabolism in plant and algal cells. Annual Review of Plant
Physiology and Plant Molecular Biology 45, 577–607.
Huppe H.C., Vanlerberghe G.C. & Turpin D.H. (1992) Evidence for
activation of the oxidative pentose phosphate pathway during
photosynthetic assimilation of nitrate but not ammonium by a
green alga. Plant Physiology 100, 2096–2099.
Hurry V.M., Keerberg O., Pärnik T., Gardeström P. & Öquist G.
(1995) Cold-hardening results in increased activity of enzymes
involved in carbon metabolism in leaves of winter rye (Secale
cereale L.). Planta 195, 554–562.
Hurry V.M., Malmberg G., Gardeström P. & Öquist G. (1994)
Effects of a short-term shift to low temperature and of long-term
cold hardening on photosynthesis and ribulose-1,5-bisphosphate
carboxylase-oxygenase activity in leaves of winter rye (Secale
cereale L.). Plant Physiology 106, 983–990.
Idso S.B. & Kimball B.A. (1992) Effects of atmospheric CO2
enrichment on photosynthesis, respiration and growth of sour
orange trees. Plant Physiology 99, 341–343.
Imsande J. & Touraine B. (1994) Nitrogen demand and the regula-
tion of nitrate uptake. Plant Physiology 105, 3–7.
Ingestad T. (1997) A shift of paradigm is needed in plant science.
Physiologia Plantarum 101, 446–450.
Jackson W.A., Flesher D. & Hageman R.H. (1973) Nitrate uptake
by dark-grown corn seedlings: some characteristics of apparent
induction. Plant Physiology 51, 120–127.
Jackson R.B. & Reynolds H.L. (1996) Nitrate and ammonium
uptake for single and mixed-species communities grown at ele-
vated carbon dioxide. Oecologia 105, 74–80.
Jacob J., Greitner C. & Drake B.G. (1995) Acclimation of photo-
synthesis in relation to Rubisco and non-structural carbohydrate
contents and in situ carboxylase activity in Scirpus olneyi grown
at elevated carbon dioxide in the field. Plant, Cell and
Environment 18, 875–884.
Jang J.C. & Sheen J. (1994) Sugar sensing in higher plants. Plant
Cell 6, 1665–1679.
Jang J.C. & Sheen J. (1997) Sugar sensing in higher plants. Trends
in Plant Science 2, 208–214.
Jang J.C., Leon P., Zhou L. & Sheen J. (1997) Hexokinase as a
sugar sensor in higher plants. Plant Cell 9, 5–19.
Jefferson R.A., Goldsbrough A. & Bevan M. (1990) Transcriptional
regulation of a patatin-1 gene in potato. Plant Molecular Biology
14, 995–1006.
Johnson D.W., Ball T. & Walker R.F. (1995) Effects of elevated car-
bon dioxide on nutrient uptake in ponderosa pine seedlings.
Plant and Soil 168, 535–545.
Kaiser W.M. & Brendle-Behnisch E. (1991) Rapid modulation of
spinach leaf nitrate reductase by photosynthesis. I. Modulation in
vivo by CO2 availability. Plant Physiology 96, 363–367.
Kaiser W.M. & Förster J. (1989) Low CO2 prevents nitrate reduc-
tion in leaves. Plant Physiology 91, 970–974.
Kaiser W.M. & Huber S.C. (1994a) Posttranslational regulation of
nitrate reductase in higher plants. Plant Physiology 106, 817–821.
Kaiser W.M. & Huber S.C. (1997) Correlation between apparent
activation state of nitrate reductase (NR), NR hysteresis and
degradation of NR protein. Journal of Experimental Botany
48, 1367–1374.
Kaiser M. & Sawers G. (1995) Nitrate repression of the Escherichia
coli pfl operon is mediated by the dual sensors NarQ an NarX
and the dual regulators NarL and NarP. Journal of Bacteriology
177, 3647–3655.
Kamberov E.S., Atkinson M.R., Chandran P. & Ninfa A.J. (1994)
Effect of mutations in Escherichia coli glnL (ntrB), encoding
nitrogen regulator II (NRII or NtrB), on the phosphatase activity
involved in bacterial nitrogen regulation. Journal of Biological
Chemistry 269, 28294–28299.
Kamberov E.S., Atkinson M.R. & Ninfa A.J. (1995) The
Escherichia coli PII signal transduction protein is activated upon
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 615
binding 2-ketoglutarate and ATP. Journal of Biological
Chemistry 270, 17797–17807.
King B.J., Siddiqi Y.M., Ruth T., Warner R.L. & Glass A.D.M.
(1993) Feedback regulation of nitrate influx in barley roots by
nitrate, nitrite and ammonium. Plant Physiology 102,
1279–1286.
King J.S., Thomas R.B. & Strain B.R. (1996) Growth and carbon
accumulation in root systems of Pinus taeda and Pinus pon-
derosa seedlings as affected by varying carbon dioxide, tempera-
ture and nitrogen. Tree Physiology 16, 635–642.
Kinsman E.-A., Lewis C., Davies M.-S., Young J.E., Francis D.,
Thomas I.-D., Chorlton K.-H. & Ougham H.-J. (1997) Effects
of temperature and elevated CO2 on cell division in shoot
meristems: Differential responses of two natural populations
of Dactylis glomerata L. Plant, Cell and Environment 19,
775–780.
Klein D. & Stitt M. (1998) Effects of 2-deoxyglucose on the expres-
sion of rbcS and the metabolism of Chenopodium rubrum cell
suspension cultures. Planta 205, 223–235.
Koch. K.E. (1996) Carbohydrate-modulated gene expression in
plants. Annual Review of Plant Physiology and Plant Molecular
Biology 47, 509–540.
Körner C. (1995) Towards a better experimental basis for upscaling
plant responses to elevated carbon dioxide and climate warming.
Plant, Cell and Environment 18, 1101–1110.
Körner C. & Miglietta F. (1994) Long term effects of naturally ele-
vated carbon dioxide on Mediterranean grassland and forest
trees. Oecologia 99, 343–351.
Körner C., Diemer M., Schappi B., Niklaus P. & Arnone J. (1997)
The responses of alpine grassland to four seasons of carbon diox-
ide enrichment: a synthesis. Acta Oecologia – International
Journal of Ecology 18, 165–175.
Körner C. Pelaez-Riedl S. & Van Bel A.J.E. (1995) CO2 responsive-
ness of plants: a possible link to phloem loading. Plant, Cell and
Environment 18, 595–600.
Kovtun Y. & Daie J. (1995) End-product control of carbon
metabolism in culture-grown sugar beet plants: molecular and
physiological evidence on accelerated leaf development and
enhanced gene expression. Plant Physiology 108, 1647–1656.
Krapp A., Fraisier V., Scheible W.-R., Quesada A., Gojon A.,
Stitt M., Caboche M. & Daniel-Vedele F. (1998) Expression
studies of Nrt2: 1Np, a putative high affinity nitrate trans-
porter: evidence for ist role in nitrate uptake. Plant Journal 6,
723–732.
Krapp A. & Stitt M. (1995) An evaluation of direct and indirect
mechanisms for the ‘sink’ regulation of photosynthesis in
spinach: changes in gas exchange, metabolites, enzyme activities
and steady state transcript levels after cold-girdling spinach
leaves. Planta 195, 313–323.
Krapp A., Quick W.P. & Stitt M. (1991) Ribulose-1, 5-bisphosphate
carboxylase-oxygenase, other Calvin cycle enzymes, and chloro-
phyll decrease when glucose is supplied to mature spinach leaves
via the transpiration stream. Planta 186, 58–69.
Krapp A., Hofmann B., Schäfer C. & Stitt M. (1993) Regulation of
the expression of rbcS and other photosynthetic genes by carbo-
hydrates: a mechanism for the ‘sink’ regulation of photosynthe-
sis? Plant Journal 3, 817–828.
Kronenberger J., Lepingle A., Caboche M. & Vaucheret H. (1993)
Cloning and expression of distinct nitrite reductases in tobacco
leaves and roots. Molecular and General Genetics 236,
203–208.
Kuiper D., Kuiper P.J.C., Lambers H., Schuit J. & Staal M. (1989)
Cytokinin concentration in relation to mineral nutrition and ben-
zyladenine treatment in Plantago major spp. pleiosperma.
Physiologia Plantarum 75, 511–517.
Kutik J., Natr L., Demmers-Derks H.H. & Lawlor D.W. (1995)
Chloroplast ultrastructure of sugar beet culitvated in normal and
616 M. Stitt and A. Krapp
elevated carbon dioxide concentrations with two contrasted nitro-
gen supplies. Journal of Experimental Botany 46, 1797–1802.
Lainé P., Ourry A. & Boucaud J. (1995) Shoot control of nitrate
uptake rates by roots of Brassica napus L. effects of localized
nitrate supply. Planta 196, 77–83.
Lam H.M., Coshigano K., Oliveira I., Melo-Oliveira R. & Coruzzi
G. (1996) The molecular genetics of nitrogen assimilation into
amino acids in higher plants. Annual Review of Plant Physiology
and Plant Molecular Biology 47, 569–593.
Lambers H., Cambridge M.L., Konings H. & Pons T.L. (1990) Causes
and Consequences of Variaton in Growth Rate and Productivity of
Higher Plants. SPB Academic Publishing bv., The Hague.
Larigauderie A., Reynolds J.F. & Strain B.R. (1994) Root response
to carbon dioxide enrichment and nitrogen supply in loblolly
pine. Plant and Soil 165, 21–32.
Lauerer M. (1996) Wachstum, Kohlenstoff- und Stickstoffhaushalt
von Nicotiana tabacum mit reduzierter Nitratreduktaseaktivität.
Dissertation. Universität Bayreuth Bayreuther Forum Ökologie,
Band 31, Bayreuther Institut für Terrestrische Ökosystem-
forschung (Hrsg.).
Lauerer M., Saftic D., Quick W.P., Labate C., Fichtner K., Schulze
E.-D., Rodermel S., Bogorad L. & Stitt M. (1993) Decreased
ribulose-1,5-bisphosphate carboxylase-oxygenase in trans-
genic tobacco transformed with ‘antisense’ rbcS. VI. Effect on
photosynthesis in plants grown at different irradiance. Planta
190, 332–345.
Lauter F.R., Ninnemann O., Bucher M., Riesmeier J.W. & Frommer
W.B. (1996) Preferential expression of an ammonium transport
and of two putative nitrate transporters in root hair of tomato.
Proceedings of the National Academy of Science of the USA 93,
8139–8144.
Lea P. & Forde B. (1994) Studies of amino acid biosynthesis in
transgenic plants. Plant, Cell and Environment 17, 541–556.
Lee H.S.J. & Jarvis P.G. (1995) Trees differ from crops and from
each other in their response to increases in CO2 concentration.
Journal of Biogeography 22, 323–330.
van de Leij M., Smith S. & Miller A.J. (1998) Remobilisation of
vacuolar stored nitrate in barley root cells. Planta 205, 64–72.
Leport L., Kandlbinder A., Baur B. & Kaiser W.M. (1996) Diurnal
modulation of phosphoenolpyruvate carboxylation in pea leaves
and roots as related to tissue malate concentrations and to the
nitrogen source. Planta 198, 495–501.
Lerchl J., Geigenberger P., Stitt M. & Sonnewald U. (1995)
Inhibition of long distance sucrose transport by inorganic phos-
phatase can be complemented by phloem-specific expression of
cytosolic yeast-derived invertase in transgenic plants. Plant Cell
7, 259–270.
Lexa M. & Cheeseman J.M. (1997) Growth and nitrogen relations
in reciprocal grafts of wild-type and nitrate reductase-deficient
mutants of pea. Journal of Experimental Botany 48, 1241–1250.
Li B., Zhang X.-Q. & Chollet R. (1996) Phosphoenolpyruvate
carboxylase kinase in tobacco leaves is activated by light in a
similar but not identical way as in maize. Plant Physiology
111, 497–505.
Lin Y., Hwang C.F., Brown J.B. & Cheng C.L. (1994) 5′ proximal
regions of Arabidopsis nitrate reductase genes direct nitrate-
induced transcription in transgenic tobacco. Plant Physiology
106, 477–484.
Lin J.T. & Stewart V. (1996) Nitrate- and nitrite-mediated transcrip-
tion antitermination control of nasF (nitrate assimilation) operon
expression on Klebsiella pneumoniae M5a1. Journal of
Molecular Biology 256, 423–435.
Lloyd J. & Farquhar G.D. (1996) The CO2 dependence of photosyn-
thesis, plant growth responses to elevated atmospheric CO2 con-
centrations and their interaction with soil nutrient status. I General
principles and forest ecosystems. Functional Ecology 10, 4–32.
Ludewig F., Sonnewald U., Kauder F., Heineke D., Geiger M., Stitt
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
M., Müller-Röber B.T., Gillissen B., Kühn C. & Frommer W.B.
(1998) The role of transient starch in acclimation to elevated
atmospheric CO2. FEBS Letters 429, 147–151.
Luo Y., Field C.B. & Mooney H.A. (1994) Predicting responses of
photosynthesis and root fraction to elevated carbon dioxide.
Interactions among carbon, nitrogen and growth. Plant, Cell and
Environment 17, 1195–1204.
MacKintosh C. (1998) Regulation of cytosolic enzymes in primary
metabolism by reversible protein phosphorylation. Current
Topics in Plant Science 1, 224–229.
Maevskaya S.N., Andreeva T.F., Voedudskaya S.Y.U. &
Cherkanova S. (1990) Effect of high carbon dioxide concentra-
tion on photosynthesis and nitrogen metabolism in leaf mustard
plants. Fiziologia Rastenii 37, 921–927.
Makino A., Nakana H. & Mae T. (1994) Responses of ribulose-1,5-
biphosphate decarboxylase, cytochrome f, and sucrose synthesis
in rice leaves to leaf nitrogen and their relationship to photosyn-
thesis. Plant Physiology 105, 173–179.
Makino A., Sakashita H., Hidema J., Mae T., Ojima K. & Osmond
B. (1992) Distinctive responses of ribulose-1,5-biphosphate
decarboxylase and carbonic anhydrase in wheat leaves to nitro-
gen nutrition and their possible relationships to CO2 transfer
resistance. Plant Physiology 100, 1737–1743.
Makino A., Shimada T., Takumi S., Kaneko K., Matsuoka M.,
Shimamoto K., Nakano H., Miyao-Tokutomi M., Mae T. &
Yamamoto N. (1997) Does decrease in ribulose-1,5-bisphos-
phate carboxylase by antisense RbcS lead to a higher N-use effi-
ciency of photosynthesis under conditions of saturating CO2 and
light in rice plants? Plant Physiology 114, 483–491.
Manh C.T., Bismuth E., Boutin J.P., Provot M. & Champigny M.-L.
(1993) Metabolite effectors for short-term enhancement of phos-
phoenolpyruvate carboxylase activity and decrease of net sucrose
synthesis in wheat leaves. Physiologia Plantarum 89, 460–466.
Marschner M. (1995) Mineral Nutrition of Higher Plants. 2nd edn.
Academic Press Limited, London, UK.
Martin T., Hellmann H., Schmidt R., Willmitzer L. & Frommer
W.B. (1997) Identification of mutants in metabolically regulated
gene expression. Plant Journal 11, 53–62.
Martinoia E. & Rentsch D. (1994) Malate compartmentation –
responses to a complex metabolism. Annual Review Plant
Physiology and Plant Molecular Biology 45, 447–467.
Marzluf G.A. (1993a) Regulation of sulfur and nitrogen metabolism
in filamentous fungi. Annual Review of Microbiology 47, 31–55.
Marzluf G.A. (1993b) Genetic regulation of nitrogen metabolism in
fungi. Microbiological and Molecular Biology Reviews 61, 1–17.
Masle J., Hudson G.S. & Badger M. (1993) Effects of ambient car-
bon dioxide concentrations on growth and nitrogen use in
tobacco plants transformed with an antisense gene to the small
subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase.
Plant Physiology 103, 1075–1088.
Matsumara T., Sakaibara H., Nakano R., Kimata Y., Sugiyama T. &
Hase T. (1997) A nitrate-inducible ferreedoxin in maize roots:
genomic organisation and differential expression of two non-
photosynthetic ferredoxin apoproteins. Plant Physiology 114,
653–660.
Matt P., Schurr U., Krapp A. & Stitt M. (1998) Growth of tobacco in
short day conditions leads to high starch, low sugars, altered diur-
nal changes of the nia transcript and low nitrate reductase activity,
and an inhibition of amino acid synthesis. Planta 207, 27–41.
Mattana M., Brambilla J., Bertani A. & Reggiani R. (1996)
Expression of nitrogen assimilating enzymes in germinating rice
under anoxia. Plant Physiology and Biochemistry 34, 653–657.
Mauri I., Maddoloni M., Lohmer S., Motto M., Salamini F.,
Thompson R. & Martegani E. (1993) Functional expression of
the transcriptional activator Opaque-2 of Zea mays in trans-
formed yeast. Molecular and General Genetics 241, 319–326.
McKee I.F. & Woodward F.I. (1994) CO2 enrichment responses of

wheat: interactions with temperature, nitrate and phosphate. New
Phytology 127, 447–453.
Meyer zu Hoerste G. (1998) Einfluss von Aminosäuren auf das
Wachstum und die Physiologie von Arabidopsis thaliana.
Diplomarbeit, Universität Heidelberg.
Micallef B.J., Haskins K.A., Vanderweer P.J., Roh K.S.,
Shrewmaker C.K. & Sharkey T.D. (1995) Altered photosynthe-
sis, flowering and fruiting in transgenic tomato plants that have
increased capacity for sucrose synthesis. Planta 196, 327–334.
Migge A., Carroyol E., Kunz C., Hirel B., Fock H. & Becker T.
(1997) The expression of the tobacco genes encoding plastidic
glutamine synthetase or ferredoxin-dependent glutamate syn-
thase does not depend on the rate of nitrate reduction, and is
unaffected by suppression of photorespiration. Journal of
Experimental Botany 48, 1175–1184.
Miglietta F., Giuntoli A. & Bindi M. (1996) The effect of free air
carbon dioxide enrichment and soil nitrogen availability on the
photosynthetic capacity of wheat. Photosynthesis Research 47,
281–290.
Miller A.J. & Smith S.J. (1996) Nitrate transport and compartmen-
tation in cereal root cells. Journal of Experimental Botany 47,
843–854.
Miller A., Tsai C.-H., Hemphill D., Endres M., Rodermel S. &
Spalding M. (1997) Elevated carbon dioxide effects during leaf
ontogeny. Plant Physiology 115, 1195–1200.
Mita S., Murano N., Akaike M. & Nakamura K. (1997) Mutants of
Arabidopsis thaliana with pleiotropic effects on the expression
of the gene for beta-amylase and on the accumulation of antho-
cyanin that are inducible by sugars. Plant Journal 11, 841–851.
Mitchell R.A.C., Mitchell V.J., Driscoll S.P., Franklin J. &
Lawlor D.W. (1993) Effects of increase carbon dioxide con-
centration and temperature on gropwth and yield of winter
wheat at two levels of nitrogen application. Plant, Cell and
Environment 16, 521–529.
Moore B.D., Cheng S.-H., Rice J. & Seeman J. (1998) Sucrose
cycling, Rubisco expression and prediction of photosynthetic
acclimation to elevated carbon dioxide. Plant, Cell and
Environment 21, 905–915.
Moore B.D., Palmquist D.E. & Seemann J.R. (1997) Influence of
plant growth at high CO2 concentrations on leaf content of ribu-
lose-1,5-bisphosphate carboxylase/oxygenase and intracellular
distribution of soluble carbohydrates in tobacco, snapdragon,
and parsley. Plant Physiology 115, 241–248.
Moorhead G., Douglas P., Morrice N., Scarabel M., Aitken A. &
MacKintosh C. (1996) Phosphorylated nitrate reductase from
spinach leaves is inhibited by 14–3–3 proteins and activated by
fusicoccin. Current Biology 6, 1104–1113.
Morcuende R., Krapp A., Hurry V. & Stitt M. (1998) Sucrose feed-
ing leads to increased rates of nitrate assimilation, increased rates
of oxoglutarate synthesis, and increased synthesis of a wide spec-
trum of amino acids in tobacco leaves. Planta 206, 394–409.
Morcuende R., Pérez P. & Martínez-Carrasco R. (1997) Short-
term feedback inhibition of photosynthesis in wheat leaves
supplied with sucrose and glycerol at two temperatures.
Photosynthetica 33, 179–188.
Morcuende R., Pérez P., Martínez-Carrasco R., Del-Molino I.M. &
Sanchez de la Puenta L. (1996) Long and short term responses of
leaf carbohydrate levels and photosynthesis to decreased sink
demand in soybean. Plant, Cell and Environment 19, 976–982.
Morin F., Andre M. & Betsche T. (1992) Growth kinetics, carbohy-
drate and leaf phosphate content of clover after transfer to a high
carbon dioxide atmosphere or to high light and ambient air. Plant
Physiology 99, 89–95.
Muller B. & Touraine B. (1993) Inhibition of nitrate uptake by vari-
ous phloem-translocated amino acids in soybean seedlings.
Journal of Experimental Botany 63, 617–623.
Müller-Röber B.T., Koßmann J., Hannah L.C., Willmitzer L. &
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 617
Sonnewald U. (1990) One of two different ADP-glucose
pyrophosphorylase genes from potato responds strongly to
elevated levels of sucrose. Molecular and General Genetics
224, 136–146.
Navarro M.T., Prieto R., Fernandez E. & Galvan A. (1996)
Constitutive expression of nitrate reductase changes the regula-
tion of nitrate and nitrate transporters in Chlamydomonas rein-
hardtii. Plant Journal 9, 819–827.
Neuhaus H.E. & Stitt M. (1990) Control analysis of photosynthate
partitioning: impact of reduced activity of ADP glucose
pyrophosphorylase on plastid phosphoglucomutase on the fluxes
to starch and sucrose in Arabidopsis thaliana L. Heynh. Planta
182, 445–454.
Newberry R.M., Wolfenden J., Mansfield T.A. & Harrison A.F.
(1995) Nitrogen, phosphorus and potassium demand in Agrostis
capillaris: the influence of elevated carbon dioxide and nutrient
supply. New Phytologist 130, 565–574.
Nie G.Y., Hendrix D.L., Webber A.N., Kimball B.A. & Long S.P.
(1995b) Increased accumulation of carbohydrates and decreased
photosynthetic gene transcript levels in wheat grown at an ele-
vated carbon dioxide concentration in the field. Plant Physiology
108, 975–983.
Nie G.Y., Long S.P., Garcia R.L., Kimball B.A., Lamorte R.L.,
Pinter P.J., Wall G.W. & Webber A.N. (1995a) Effects of free-air
CO2 enrichment on the development of the photosynthetic appa-
ratus in wheat, as indicated by changes in leaf proteins. Plant,
Cell and Environment 18, 855–864.
Nielsen T.H., Krapp A., Röper-Schwarz U. & Stitt M. (1998) The
sugar-mediated regulation of genes encoding the small subunit of
Rubisco and the regulatory subunit of ADP glucose pyrophos-
phorylase is modified by phosphate and nitrogen. Plant, Cell and
Environment 21, 443–454.
Ninneman O., Jauniaux J.-C. & Frommer W.B. (1994)
Identification of a high afffinity ammonium transporter from
plants. EMBO Journal 13, 3464–3471.
Norby R.J., O’Niell E.G. & Luxmore R.J. (1986) Effects of atmo-
spheric CO2 enrichment on growth and mineral nutrition of
Quercus alba seedlings in a nutrient poor soil. Plant
Physiology 82, 83–89.
Nussaume L., Vincentz M., Meyer C., Boutin J.P. & Caboche M.
(1995) Post-transcriptional regulation of nitrate reductase by
light is abolished by a N-terminal deletion. Plant Cell 7,
611–621.
O’Niell E.G., Lumore R.J. & Norby R.J. (1987) Elevated CO 2
effects on seedling growth, nutient uptake, and rhizosphere
bacterial populations of Liriodendron tulipifera L. Plant and
Soil 104, 3–11.
Oberbauer S.F., Sionit N., Hastings S.J. & Oechel W.C. (1986)
Effects of CO2 enrichment and nutrition on growth, photosynthe-
sis and nutrient concentration of Alaskan tundra plant species.
Canadian Journal of Botany 64, 2993–2998.
Oechel E.C., Hastings J., Vourlitis M., Jenkins M., Riechers G. &
Grulke N. (1993) Recent change of artic tundra ecosystems from
a net carbon dioxide sink to a source. Nature 361, 520–523.
Oparka K., Viola R., Wright K.M. & Prior D.A.M. (1992) Sugar
transport and metabolism in the tuber. In Carbon Partitioning
Within and Between Organisms (eds. C.J. Pollock, J.F. Farrar &
A.J. Gordon), pp. 1–26. BIOS Scientific Publishers Limited,
Oxford.
Özcan S., Dover J., Rosenwald A.G., Woelf S. & Johnston M.
(1996) Two glucose transporters in Saccharomyces cerevisiae
are glucose sensors that generate a signal for induction of gene
expression. Proceedings of the National Academy of Science of
the USA 93, 12428–12428.
Pain V.M. (1994) Translational control during amino acid starva-
tion. Biochemie (Paris) 76, 718–728.
Pajuelo P., Pajuelo E., Forde B.G. & Marquez A.J. (1997)
618 M. Stitt and A. Krapp
Regulation of the expression of ferredoxin glutamate synthase in
barley. Planta 203, 517–525.
Paul M. & Driscoll S.P. (1997) Sugar repression of photosynthesis
during N deficiency. Plant, Cell Environment 20, 110–116.
Paul M. & Stitt M. (1993) Effects of nitrogen and phosphate defi-
ciencies on levels of carbohydrates, respiratory enzymes and
metabolites in seedlings of tobacco, and their response to exoge-
nous sucrose. Plant, Cell and Environment 16, 1047–1057.
Pearson M. & Brooks G.L. (1995) The influence of elevated carbon
dioxide on growth and age-related changes in leaf gas exchange.
Journal of Experimental Botany 46, 1651–1659.
Peng J., Carol P., Richards D.E., King K.E., Cowling R., Murphy
G.P. & Harberd N.P. (1997) The Arabidopsis GAI gene defines a
signaling pathway that negatively regulates gibberillin
responses. Genes and Development 11, 3194–3205.
Peters W., Füchtbauer B. & Beck E. (1995) Nitrate reductase activ-
ity is endogenously induced by zeatin riboside in habituated sus-
pension cultured Chenopodium rubrum cells. Journal of Plant
Physiology 147, 401–407.
Pettersson R. & MacDonald J.S. (1994) Effects of nitrogen supply
on the acclimation of photosynthesis to elevated CO2.
Photosynthesis Research 39, 389–400.
Pettersson R., MacDonald J.S. & Stadenburg I. (1993) Response of
small birch plants (Betula pendula Roth.) to elevated CO2 and
nitrogen supply. Plant, Cell and Environment 16, 1115–1121.
Poethig R.S. (1990) Phase change and the regulation of shoot mor-
phogenesis in plants. Science 250, 923–930.
Poorter H., Berkel V., Baxter R., den Hertog J., Dijkstra P., Gifford
R.M., Griffin K.L., Rounet C., Roy J. & Wong S.C. (1997) The
effect of elevated carbon dioxide on the chemical composition
and construction costs of leaves of 27, C3 species. Plant, Cell
and Environment 20, 472–482.
Pouteau S., Cherel I., Vaucheret H. & Caboche M. (1989) Nitrate
reductase mRNA regulation in Nicotiana plumbaginifolia nitrate
reductase-deficient mutants. Plant Cell 1, 1111–1120.
Preiss J. (1991) Biology and molecular biology of starch synthesis
and its regulation. In Oxford Surveys of Plant Molecular and
Cell Biology (eds. B. Miflin) Vol. 7, pp. 59–114. Academic
Press, New York.
Pritchard S.G., Peterson C.M., Prior S.A. & Rogers H.H. (1997)
Elevated carbon dioxide differentially affects needle ultra-
structire and phloem anatomy in Pinus palustris: interactions
with soil resource availability. Plant, Cell and Environment
20, 461–471.
Prieto R., Dubus A., Galvan A. & Fernandez E. (1996) Isolation and
characterisation of two new regulatory mutante for nitrate assimi-
lation in Chlamydomonas reinhardtii obtained by insertional
mutagenesis. Molecular and General Genetics 251, 461–471.
Purvis A.C., Peters D.B. & Hageman R.H. (1974) Effect of carbon
dioxide on nitrate accumulation and nitrate reductase induction
in corn seedlings. Plant Physiology 53, 934–941.
Quesada A., Galvan A. & Fernandez E. (1994) Identification of
nitrate transporter genes in Chlamydomonas reinhardtii. Plant
Journal 5, 407–419.
Quesada A., Krapp A., Trueman L.J., Daniel-Vedele F., Fernandez
E., Forde B. & Caboche M. (1997) PCR-identification of a
Nicotiana plumbaginifolia cDNA homologous to the high-affin-
ity nitrate transporter of the crnA family. Plant Molecular
Biology 34, 265–274.
Quesada. A., Galvan A., Schnell R., Lefebvre P.A. & Fernandez E.
(1993) Five nitrate assimilation loci are clustered in
Chlamydomonas reinhardtii. Molecular and General Genetics
237, 429–437.
Quick W.P., Fichtner K., Schulze E.-D., Wendler R., Leegood R.,
Rodermel S.R., Bogorad L. & Stitt M. (1993) Decreased ribu-
lose-1,5-bisphosphate carboxylase-oxygenase in transgenic
tobacco transformed with antisense rbcS. IV Impact on photo-
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
synthesis in conditions of altered nitrogen supply. Planta 188,
522–531.
Radoglou K.M., Aphalo P. & Jarvis P.G. (1992) Response of photo-
synthesis, stomatal conductance and water use efficiency toele-
vated carbon dioxide and nutrient supply in acclimated seedlings
of Phaseolus vulgaris L. Annals of Botany 70, 257–264.
Rao M. & Terry N. (1989) Leaf phosphate status, photosynthesis,
and carbon partitioning in sugar beet: I Changes in growth, gas
exchange and Calvin cycle enzymes. Plant Physiology 90,
814–819.
Reddy A.R., Reddy K.R., Padjung R. & Hodges H.F. (1996)
Nitrogen nutrition and photosynthesis in leaves of pima cotton.
Journal of Plant Nutrition 19, 755–770.
Redinbaugh M.G. & Campbell W.M. (1991) Higher plant responses
to environmental nitrate. Physiologia Plantarum 82, 640–650.
Redinbaugh M.G. & Campbell W.H. (1993) Glutamine synthetase
and ferredoxin-dependent glutamate synthase expression in the
maize (Zea mays) root: primary response to nitrate. Plant
Physiology 101, 1249–1255.
Rentsch D., Boorer K. & Frommer W.B. (1998) Molecular biology
of sucrose, amino acid and oligopeptide transporters at the plas-
malemma membrane of plant cells. Journal of Membrane
Biology 162, 177–180.
Riesmeier J., Willmitzer L. & Frommer W.B. (1994) Antisense
repression of sucrose transporter affects assimilate partitioning in
transgenic potato plants. EMBO Journal 13, 1–7.
Ritchie S.W., Redinbaugh M.G., Shiraishi N., Verba J.M. &
Campbell W.H. (1994) Identification of a maize root transcript
expressed in the primary response to nitrate: characterization of a
cDNA with homology to ferredoxin-NADP (+) oxidoreductase.
Plant Molecular Biology 26, 679–690.
Riviere-Rolland H., Contard P. & Betsche T. (1996) Adaptation of
pea to elevated atmospheric CO 2: Rubisco, PEP carboxylase
and chloroplast phosphate translocator at different levels of
nitrogen and phosphate nutrition. Plant, Cell and Environment
19, 109–117.
Rogers G.S., Milham P.J., Gillings M. & Conroy J.P. (1996b) Sink
strength may be the key to growth and nitrogen response in N-
deficient wheat at elevated carbon dioxide. Australian Journal of
Plant Physiology 23, 253–264.
Rogers G.S., Payne L., Milham P. & Conroy J. (1993) Nitrogen and
phosphorus requirements of cotton and wheat under changing
CO2 concentrations. Plant and Soil 155, 231–234.
Roitsch T., Büttner M. & Godt D.E. (1995) Induction of apoplastic
invertase of Chenopodium rubrum by D-glucose and a glucose
analogue and tissue specific expression suggest a role in source-
sink regulation. Plant Physiology 108, 285–294.
Rook F., Weisbeek P. & Smeekens S. (1998) The light-regulated
Arabidopsis bZIP transfactor gene ATB2 encodes a protein with
an unusually long leucine zipper domain. Plant Molecular
Biology 37, 171–178.
Ross H.A., Davies H.V., Burch L.R., Violoa R. & McRae D. (1994)
Developmental changes in carbohydrate content and sucrose
degrading enzymes in tuberising stolons of potato. Physiologia
Plantarum 90, 748.756.
Roumet C., Bel M.P., Sonie L., Jardon F. & Roy J. (1996) Growth
response of grasses to elevated carbon dioxide: a physiological
plurispecific response. New Phytologist 133, 595–603.
Rufty T.W. Jr, Huber S.C. & Volk R.J. (1988) Alterations in leaf car-
bohydrate metabolism in response to nitrogen stress. Plant
Physiology 88, 725–730.
Rufty T.W. Jr, Israel D.W., Volk R.J., Qiu J. & Sa T. (1993)
Phosphate regulation of nitrate assimilation in soybean. Journal
of Experimental Botany 44, 879–891. 1993.
Sage R.F. (1994) Acclimation of photosynthesis to increasing atmo-
spheric carbon dioxide; the gas exchange perspective.
Photosynthesis Research 39, 351–368.

Sage R.F., Sharkey T.D. & Seemann J.R. (1990) Regulation of ribu-
lose-1,5-bisphosphate carboxylase activity in response to light
intensity and CO2 in the C3 annuals Chenopodium album L. &
Phaseolus vulgaris L. Plant Physiology 94, 1735–1742.
Sakakibara H., Suzuki M., Takei M., Deji A., Taniguchi M. &
Sugiyama T. (1998) A response-regulator homolog possibly
involved in nitrogen signal transduction mediated by cytokinin in
maize. Plant Journal 14, 337–344.
Sakakibara. H., Watanabe M., Hase T. & Sugiyama T. (1991)
Molecular cloning and characterisation of complementary DNA
encoding for ferredoxin-dependent glutamate synthase in maize
leaf. Journal of Biological Chemistry 266, 2028–2035.
Samuelson M.E., Campbell W.H. & Larsson C.M. (1995) The
influence of cytokinin in nitrate regulation of nitrate reductase
activity and expression in barley. Physiologia Plantarum 93,
533–539.
Samuelson M.E., Eliason L. & Larsson C.M. (1992) Nitrate-regu-
lated growth and cytokinin responses in seminal roots of barley.
Plant Physiology 98, 309–315.
Sattelmacher B. & Thoms K. (1995) Morphology and physiology of
the seminal root system of young maize plants as influenced by a
locally restricted nitrate supply. Zeitung Fuer Pflanzenernährung
Bodenkunde 158, 493–497.
Schappi B. & Körner C. (1997) In situ effects of elevated carbon
dioxide on the carbon and nitrogen status of alpine plants.
Functional Ecology 11, 290–299.
Scheible W.-R., Lauerer M., Schulze E.-D., Caboche M. & Stitt M.
(1997a) Accumulation of nitrate in the shoot acts as signal to regu-
late shoot-root allocation in tobacco. Plant Journal 11, 671–691.
Scheible W.-R., Gonzales-Fontes A., Lauerer M., Müller-Röber B.,
Caboche M. & Stitt M. (1997b) Nitrate acts as a signal to induce
organic acid metabolism and repress starch metabolism in
tobacco. Plant Cell 9, 783–798.
Scheible W.-R., Gonzalez-Fontes A., Morcuende R., Lauerer M.,
Geiger M., Glaab J., Gojon., Schulze E.-D. & Stitt M. (1997c)
Tobacco mutants with a decreased number of functional nia
genes compensate by modifying the diurnal regulation of tran-
scription, post-translational modification and turnover of nitrate
reductase. Planta 203, 304–319.
Schenk U., Manderschied R., Hugen J. & Weigel H.J. (1995) Effects
of CO2 enrichment and intraspecific competition on biomass
partitioning, nitrogen content and microbial biomass of perennial
white clover. Journal of Experimental Botany 46, 987–993.
Schnell R. & Lefebrve P.A. (1993) Isolation of the
Chlamydomonas regulatory gene nit-2 by transposon tagging.
Genetics 134, 737–747.
Schulze W., Schulze E.-D., Stadler J., Heilmeier H., Stitt M. &
Mooney H.A. (1994) Growth and reproduction of Arabidopsis
thaliana in relation to storage of starch and nitrate in the wildtype
and in starch-deficient and nitrate-uptake deficient mutants.
Plant, Cell and Environment 17, 795–809.
Shaner D.L. & Boyer J.S. (1976) Nitrate reductase activity in maize
(Zea mays L.) leaves. I. Regulation by nitrate flux. Plant
Physiology 58, 499–504.
Sharma A. & Sen Gupta U.K. (1990) Carbon dioxide enrichment
effects on photosynthesis and related enzymes in Vigna radiata
L. Wilczek. Indian Journal of Plant Physiology 33, 340–346.
Sheen J. (1994) Feedback control of gene-expression. Photosynthesis
Research 39, 427–438.
Sicher R.C. & Bunce J.A. (1997) Relationship of o photosynthetic
acclimation to changes of Rubisco activity in field grown winter
wheat and barley during growth in elevated carbon dioxide.
Photosynthesis Research 52, 27–38.
Sicher R.C. & Kremer D.F. (1994) Responses of Nicotiana
tabacum to carbon dioxide enrichment at low-photon flux den-
sity. Physiologia Plantarum 92, 383–388.
Sicher R.C., Kremer D.F. & Bunce J.A. (1995) Photosynthetic
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 619
acclimation and photosynthate partitioning in soybean leaves in
response to carbon dioxide enrichment. Photosynthesis Research
46, 409–417.
Sicher R.C., Kremer D.F. & Rodermel S.R. (1994) Photosynthetic
acclimation to elevated carbon dioxide occurs in transformed
tobacco with decreased ribulose-1,5-bisphosphate carboxylase/
oxygenase content. Plant Physiology 104, 409–415.
Siddiqi M.Y., Glass A.D.M., Ruth T.J. & Rufty T.W. (1990) Studies
of the uptake of nitrate in barley. Plant Physiology 93, 1426–1432.
Silverstone A.L., Ciampaglio C.N. & Sun T.P. (1998) The
Arabidopsis RGA gene encodes a transcriptional regulator
repressing the gibberillin signal transduction pathway. Plant Cell
10, 155–169.
Silvola J. & Ahlholm U. (1995) Combined effects of carbon dioxide
concentration and nutrient status on the biomass production and
nutrient uptake of birch seedlings. Plant and Soil 169, 547–553.
Sionit N. (1983) Response of soybean to two levels of mineral nutri-
tion in CO2 enriched atmosphere. Crop Science 23, 329–333.
Sivasankar S. & Oaks A. (1996) Nitrate assimilation in higher
plants: the effect of metabolites and light. Plant Physiology and
Biochemistry 34, 609–620.
Sivasankar S., Rothstein S. & Oaks A. (1997) Regulation of the
accumulation and reduction of nitrate by nitrogen and carbon
metabolites in maize seedlings. Plant Physiology 114, 583–589.
Smeekens S. (1998) Sugar regulation of gene expression in plants.
Current Opinion in Plant Biology 1, 230–234.
Smeekens S. & Rook F. (1997) Sugar sensing and sugar-mediated
signal transduction in plants. Plant Physiology 115, 7–13.
Socias F.X., Medrano H. & Sharkey T.D. (1993) Feedback limita-
tion of photosynthesis of Phaseolus vulgaris L. grown in ele-
vated carbon dioxide. Plant, Cell and Environment 16, 81–86.
Somerville C.R. (1986) Analysis of photorespiration with mutants
of higher plants and algae. Annual Review of Plant Physiology
37, 467–507.
Sonnewald U., Hajirezaei M.-R., Heyer A., Trethewey R.N. &
Willmitzer L. (1997) Expression of yeast invertase in the
apoplast of potato tubers increases tubers size. Nature
Biotechnology 15, 794–797.
Sonnewald U., von Brauer M., Schwaeren A., Stitt M. & Willmitzer
L. (1991) Transgenic tobacco plants expressing yeast-derived
invertase in either the cytosol, vacuole or apoplast: a powerful
tool for studying sucrose metabolism and sink/source interac-
tions. Plant Journal 1, 95–106.
Stewart V. (1994) Regulation of nitrate and nitrite reductase synthe-
sis in enterobacteria. Antonie Van Leeuwenhoek 66, 37–45.
Stitt M. (1991) Rising CO2 levels and their potential significance
for carbon flow in photosynthetic cells. Plant, Cell and
Environment 14, 741–762.
Stitt M. (1996) Metabolic regulation of photosynthesis. In
Advances in Photosynthesis, Vol. 5. Photosynthesis and the
Environment (ed. N.R. Baker) pp. 151–190. Kluwer Academic
Publishers, Dordrecht.
Stitt M., von Schaewen A. & Willmitzer L. (1991) Sink regulation
of photosynthetic metabolism in transgenic tobacco plants
expressing yeast invertase in their cell wall involves a decrease
of the Calvin cycle enzymes and an increase of glycolytic
enzymes. Planta 183, 40–50.
Stitt M. & Scheible W.-R. (1998) Understanding allocation to shoot
and root growth will require molecular information about which
compounds act as signals, and how meristem activity and cellular
growth are regulated: opinion. Plant Soil 201, 259–263.
Stitt M. & Scheible W.-R. (1999) Nitrate acts as a signal to control
gene expression, metabolism and biomass allocation. In
Regulation of Metabolism (ed. N. Kruger) in press.
Stitt M. & Schulze E.-D. (1994) Does Rubisco control the rate of
photosynthesis and plant growth? An exercise in molecular eco-
physiology. Plant, Cell and Environment 17, 518–552.
620 M. Stitt and A. Krapp
Stitt. M. & Feil R. (1999) Lateral roots decrease when nitrate accu-
mulates in tobacco transformants with low nitrate reductase
activity. Plant Soil in press.
Strand A., Hurry V., Gustafsson P. & Gardestrom P. (1997)
Development of Arabidopsis leaves at low temperatures releases
the suppression of photosynthesis and photosynthetic gene
expression despite accumulation of soluble carbohydrate. Plant
Journal 12, 605–614.
Sugiharto B. & Sugiyama T. (1992) Effects of nitrate and ammo-
nium on gene expression of phosphoenolpyruvate carboxylase in
maize leaf tissue during recovery from nitrogen stress. Plant
Physiology 98, 1403–1408.
Sugiharto B., Burnell J.N. & Sugiyama T. (1992a) Cytokinin is
required to induce the nitrogen-dependent accumulation of
mRNA’S for phosphoenolpyruvate carboxylase and carbonic
anhydrase in detached maize leaves. Plant Physiology 100,
153–156.
Sugiharto B., Suzuki I., Burnell J. & Sugiyama T. (1992b)
Glutamine induces the N-dependent accumulation of mRNA
encoding phosphoenolpyruvate carboxylase and carbonic anhy-
drase in detached maize leaves. Plant Physiology 100,
2066–2070.
Suzuki I., Cretin C., Omata T. & Sugiyama T. (1994)
Transcriptional and post-transcriptional regulation of nitrogen-
responding expression of phosphoenolpyruvate carboxylase
gene in maize. Plant Physiology 105, 1223–1229.
Taniguchi M., Kiba T., Sakakibara H., Ueguchi C., Mizuno T. &
Sugiyama T. (1998) Expression of Arabidopsis response regula-
tor homologs is induced by cytokinins and nitrate. FEBS Letters
429, 259–262.
Terashima I. & Evans J.R. (1988) Effects of light and nitrogen
nutrition on the photosynthetic apparatus in spinach. Plant, Cell
and Physiology 29, 143–155.
Teskey R.O. (1995) A field study of the effects of elevated CO2
on carbon assimilation, stomatal conductance and leaf and
branch growth of Pinus taeda trees. Plant, Cell and
Environment 18, 565–573.
Thomas R.B., Lewis J.D. & Strain B.R. (1994) Effects of leaf nutri-
ent status on photosynthetic capacity in loblolly pine seedlings
grown in elevated atmospheric CO2. Tree Physiology 14,
947–950.
Thomas B.R., Terashima M., Katoh S., Stolz T. & Rodriguez R.L.
(1995) Metabolic regulation of sink–source interactions in cere-
als. In Carbon Partitioning and Sink–source Interactions in
Plants (ed. M.A. Madore & W.J. Lucas), ASPP, Baltimore.
Tingey D.T., Johnson M.G., Phillips D.L., Johnson D.W. & Ball
J.T. (1996) Effects of elevated CO2 and nitrogen on the syn-
chrony of shoot and root growth in ponderosa pine. Tree
Physiology 16, 905–914.
Tissue D.T., Thomas R.B. & Strain B.R. (1993) Long-term
effects of elevated CO2 and nutrients on photosynthesis and
Rubisco in loblolly pine seedlings. Plant, Cell and
Environment 16, 859–865.
Tissue D.T., Thomas R.B., Strain B. & R. (1997) Atmospheric
CO2 enrichment increases growth and photosynthesis of Pinus
taeda, a 4 year experiment in the field. Plant, Cell and
Environment 20, 1123–1134.
Trethewey R., Geigenberger P., Riedel K., Hajirezaei M.,
Sonnewald U., Stitt M., Riesmeier J. & Willmitzer L. (1998)
Combined expression of glucokinase and invertase in potato
tubers leads to a dramatic reduction in starch synthesis and a
stimulation of glycolysis. Plant Journal 15, 109–118.
Trueman L.J., Richardson A. & Forde B.G. (1996) Molecular
cloning of higher plant homologues of the high affinity nitrate
transporters of Chlamydomonas reinhardtti and Aspergillus
nidulans. Gene 175, 223–231.
Truong H.N., Caboche M. & Daniel-Vedele F. (1997) Sequence and
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
characterization of two Arabidopsis thaliana cDNAs isolated by
functional complementation of a yeast gln3 gdh1 mutant. FEBS
Letter 410, 213–218.
Tsai C.-H., Miller A., Spalding M. & Rodermel S. (1997) Source
strength regulates an early phase transition of tobacco shoot mor-
phogenesis. Plant Physiology 115, 907–914.
Tsay Y.F., Schroeder J.I., Feldmann A. & Crawford N.M. (1993)
The herbicide sensitivity gene CHL1 of Arabidopsis encodes a
nitrate inducible nitrate transporter. Cell 72, 705–713.
Tuba Z., Szente K., Csintalan Z. & Koch J. (1996) Responses of
carbon dioxide assimilation, transpiration and water use effi-
ciency to long-term elevated carbon dioxide in perennial xeric
loess steppe species. Journal of Plant Physiology 148, 356–361.
Turpin D.H., Weger H.G. & Huppe H.C. (1997) Interactions
between photosynthesis, respiration and nitrogen assimilation. In
Plant Metabolism (eds D.T. Dennis, D.H. Turpin, D.D. Lefebvre
& D.B. Layzell), pp. 509–524. Longman, Singapore.
Ullrich W.R., Larsson M., Larsson C.H., Lesch S. & Novacky A.
(1984) Ammonium uptake in Lemna gibba G-1, related mem-
brane potential changes and inhibition of anion uptake.
Physiologia Plantarum 61, 369–376.
Unden G., Becker S., Bongaerts J., Holighoaus G., Schirawski J. &
Six S. (1995) Oxygen sensing and oxygen-dependent gene regu-
lation in facultatively anaerobic bacteria. Archives of
Microbiology 164, 81–90.
Van de Werf A. & Nagel O.W. (1996) Carbon allocation to shoots
and roots in relation to nitrogen supply is mediated by cytokinins
and sucrose. Opinion in Plant Soil 185, 21–32.
Van Der Kooij T.A.W. & De Kok L.J. (1996) Impact of elevated
CO2 on growth and development of Arabidopsis thaliana L.
Phyton (Horn) 36, 173–184.
Van Ginkel J.H., Gorissen A. & Van Veen J.A. (1997) Carbon and
nitrogen allocation in Lolium perenne in response to elevated
atmospheric CO2 with emphasis on soil carbon dynamics. Plant
and Soil 188, 299–308.
Van Oosten J.-J. & Besford R.T. (1995) Some relationships
between the gas exchange, biochemistry and molecular biology
of photosynthesis during leaf development of tomato plants after
transfer to different carbon dioxide concentrations. Plant, Cell
and Environment 18, 1253–1256.
Van Oosten J.-J. & Besford R.T. (1996) Acclimation of photosyn-
thesis to elevated carbon dioxide through feedback regulation of
gene expression: climate of opinion. Photosynthesis Research
48, 353–365.
Van Oosten J.-J., Wilkins D. & Besford R.T. (1994) Regulation of
the expression of photosynthetic nuclear genes by high CO2 is
mimicked by carbohydrates: a mechanism for the acclimation of
photosynthesis to high CO2. Plant, Cell Environment 17,
913–923.
Van Vuuren M.M.I., Robinson D., Fitter A.H., Chasalow S.D.,
Williamson L. & Raven J.A. (1997) Effects of elevated atmo-
spheric carbon dioxide and soil water availability on root
biomass, root length and N, P and K uptake in wheat. New
Phytologist 135, 455–465.
Vaucheret H., Chabaud M., Kronenberger J. & Caboche M. (1990)
Functional complementation of tobacco and Nicotiana
plumbaginifolia nitrate reductase deficient mutants by transfor-
mation with the wild-type alleles of the tobacco structural genes.
Molecular and General Genetics 220, 468–474.
Veith R. & Komor E. (1993) Regulation of growth, sucrose storage
and ion content in sugar cane cells, measured with suspension
cells in continuous culture growth under nitrogen, phosphorus or
carbon limitation. Journal of Plant Physiology 142, 414–424.
Vincentz M. & Caboche M. (1991) Constitutive expression of
nitrate reductase allows normal growth and development of
Nicotiana plumbaginifolia plants. EMBO Journal 10,
1027–1035.

Vincentz M., Moureaux T., Leydecker M.T., Vaucheret H. &
Caboche M. (1993) Regulation of nitrate and nitrite reductase
expression in Nicotiana plumbaginifolia leaves by nitrogen and
carbon metabolites. Plant Journal 3, 313–324.
Volk R.J. (1997) Unidirectional fluxes of nitrate into and out of
maize roots: measurement and regulation by prior nitrate nutri-
tion. Plant Science 123, 1–7.
Vu J.V.C., Allen L.H. Jr, Boote K.J. & Bowes G. (1997) Effects of
elevated carbon dioxide and temperature on photosynthesis and
Rubisco in rice and soybean. Plant, Cell and Environment 20,
68–76.
Wagner B.M. & Beck E. (1993) Cytokinins in the perennial herb
Urtica dioica L. as influenced by its nitrogen status. Planta 190,
511–518.
Walker D.A. & Sivak M.N. (1986) Photosynthesis and phosphate a
cellular affair? Trends in Biochemical Science 11, 176–179.
Wang M.Y., Siddiqi M.Y., Ruth T.J. & Glass A.D.M. (1993)
Ammonium uptake by rice roots. Plant Physiology 103,
1259–1267.
Waring R.H., McDonald A.J.S., Larsson S., Ericsson T., Wiren A.,
Arwidsson E., Ericsson A. & Lohammar T. (1985) Differences in
chemical composition of plants grown at constant relative
growth rates with stable mineral nutrition. Oecologia 66,
157–160.
Warner R.L. & Huffaker R.C. (1989) Nitrate transport is indepen-
dent of NADH and NAD (P) H nitrate reductases in barley
seedlings. Plant Physiology 91, 947–953.
Webber A.N., Nie G.-Y. & Long S.P. (1994) Acclimation of photo-
synthetic proteins to rising atmospheric CO2. Photosynthesis
Research 39, 413–425.
Weber H., Borisjuk L., Heim U., Buchner P. & Wobus U. (1995a)
Seed coat associated invertases of fava bean control both unload-
ing and storage functions: cloning of cDNA’s and cell type-spe-
cific expression. Plant Cell 7, 1835–1846.
Weber H., Borisjuk L., Heim U., Buchner P. & Wobus U. (1995b)
Seed coat-associated invertases of fava bean control both unload-
ing and storage functions: Cloning of cDNAs and cell type spe-
cific expression. Plant Cell 7, 1835–1846.
Weber H., Borisjuk L., Heim U., Sauer N. & Wobus U. (1997a) A
role for sugar transporteres during seed development: molecular
characterisation of a hexose and a sucrose transporter in fava
bean seeds. Plant Cell 9, 895–908.
Weber H., Borisjuk L. & Wobus U. (1997b) Sugar import and
metabolism during seed development. Trends in Plant Sciences
2, 161–174.
Webster B.D. & Radin J.W. (1972) Growth and development of cul-
tured radish tips. American Journal of Botany 59, 744–751.
Wenzler H.C., Mignery G.A., Fisher L.M. & Park W.D. (1989)
Analysis of a chimeric class-I paptatin-GUS gene in transgenic
© 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 583–621
Elevated carbon dioxide and nitrogen nutrition 621
potato plants: high-level expression in tubers and sucrose-
inducible expression in cultured leaf and stem explants. Plant
Molecular Biology 12, 41–50.
Whitehead S.J., Caporn S.J.M. & Press M.C. (1997) Effects of ele-
vated carbon dioxide, nitrogen and phosphorus on the growth
and photosynthesis of two upland perennial. New Phytologist
135, 201–211.
Wong S.C. (1979) Elevated atmospheric partial pressure of CO2
and plant growth. I Interaction of nitrogen nutrition and photo-
synthetic capacity in C3 and C4 plants. Oecologia 44, 68–74.
Wong S.C. (1990) Elevated atmospheric partial pressure of carbon
dioxide and plant growth. II Nonstructural carbohydrate content
in cotton plants and effect on growth parameters. Photosynthesis
Research 23, 171–180.
Wong S.C. & Osmond C.B. (1991) Elevated atmospheric partial
pressure of CO2 and plant growth. III Interactions between
Triticum aestivum (C3) and Echinochlea frumentacea (C4) dur-
ing growth in mixed culture under different CO2, N nutrition and
irradiance treatments, with emphasis on below ground responses
estimated using the δ-13C value of root biomass. Australian
Journal of Plant Physiology 18, 137–152.
Wong S.C., Kriedemann P.E. & Farquhar G. (1992) Carbon diox-
ide–nitrogen interaction on seedling growth of four species of
eucalypt. Australian Journal of Botany 40, 457–472.
Woodrow I.E. (1994) Control of steady-state photosynthesis in sun-
flowers growing in enhanced CO2. Plant, Cell and Environment
17, 277–286.
Wray J.L. (1993) Molecular biology, genetics and regulation of
nitrite reduction in higher plants. Physiologia Plantarum 89,
607–612.
Xiao X., Fu Y.H. & Marzluf G.A. (1995) The negative-acting NMR
regulatory protein of Neurospora crassa binds to and inhibits the
DNA-binding activity of the positive-acting nitrogen regulatory
protein NIT2. Biochemistry 34, 8861–8868.
Yelle S., Gosselin A. & Trudel M.J. (1987) Effect of atmospheric CO2
concentration and root-zone temperature on growth, mineral nutri-
tion and nitrate reductase activity of greenhouse tomato. Journal of
the American Society of Horticultural Science 10, 36–40.
Yokoyama R., Hirose T., Fujii N., Aspuria E.T., Kato A. &
Uchimiya H. (1994) The rolC promoter of Agrobacterium rhizo-
genes Ri plasmid is activated by sucrose in transgenic tobacco
plants. Molecular and General Genetics 244, 15–22.
Zhang H. & Forde B.G. (1998) An Arabidopsis MADS-box gene
controlling nutrient-induced changes in root architecture.
Science 279, 407–409.
Ziska L.H., Weerakoon W., Namuco O.S. & Pamplona R. (1996)
The influence of nitrogen on the elevated carbon dioxide
response in field grown rice. Australian Journal of Plant
Physiology 23, 45–52.