Journal of Experimental Botany, Vol. 53, No.

370,
Inorganic Nitrogen Assimilation Special Issue, pp. 865874, April 2002

Interaction of cytosolic and plastidic

nitrogen metabolism in plants
Andreas Weber1 and Ulf-Ingo Flugge
Universitat zu Koln, Lehrstuhl Botanik II, Gyrhofstr. 15, D-50931 Koln, Germany
Received 18 July 2001; Accepted 10 December 2001

Abstract

Subcellular localization of the main ammonia

assimilation pathway in plants
In green leaves of most C3-type angiosperm plant species,
ammonia is assimilated into nitrogen compounds via
the plastidic glutamine synthetaseuglutamate synthase
(GSuGOGAT) cycle (Miflin and Lea, 1980) with the plastidic GS2 being the predominant isozyme in these tissues
(McNally et al., 1983). The plastidic GS2uGOGAT cycle
has to cope with ammonia produced by nitrateunitrite
reduction and, to a larger extent, with ammonia released
in the photorespiratory cycle. In several achlorophyllous
plant parasites, however, cytosolic GS1 is the only
detectable isozyme and in several C4-type and CAM
plants, the contribution of GS1 to total GS-activity is
about 3580% (McNally et al., 1983).
The predominant role of the GSuGOGAT-system for
the reassimilation of ammonia released during the
regeneration of 3-phosphoglycerate in the photorespiratory pathway was further established by the isolation of
mutants defective in the ferredoxin-dependent glutamate
synthase (Fd-GOGAT) and in GS2. An A. thaliana
mutant that is defective in Fd-GOGAT activity has been
described (Somerville and Ogren, 1980). This mutant
is unable to grow under ambient CO2 conditions and is
only viable under high CO2 conditions that suppress

To whom correspondence should be addressed. Fax: q49 221 4705039. E-mail: andr.weber@uni-koeln.de
Abbreviations: GS, glutamine synthetase; GOGAT, glutamine, 2-oxoglutarate aminotransferase; CAM, crassulacean acid metabolism; Fd-GOGAT,
ferredoxin-dependent GOGAT; GUS, b-glucuronidase; DiT1, dicarboxylate translocator 1 (2-oxoglutarateumalate-translocator); DiT2, dicarboxylate
trasnlocator 2 (glutamateumalate-translocator); OPPP, oxidative pentose phosphate pathway; GPT, glucose 6-phosphateuphosphate-translocator;
Glc6P, glucose 6-phosphate; NAD(P)H-GOGAT, NAD(P)H-dependent GOGAT; CitT, citrate transporter.

Society for Experimental Biology 2002

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In angiosperms, the assimilation of ammonia resulting from nitrate reduction and from photorespiration
depends on the operation of the plastidic GS/GOGAT
cycle. The precursor for ammonia assimilation, 2oxoglutarate, is synthesized in the mitochondria and
in the cytosol. It is imported into the plastid by a
2-oxoglutarate/malate translocator (DiT1). In turn,
the product of ammonia assimilation, glutamate, is
exported from the plastids by a glutamate/malate
translocator (DiT2). These transport processes link
plastidic and cytosolic nitrogen metabolism and are
essential for plant metabolism. DiT1 was purified
to homogeneity from spinach chloroplast envelope
membranes and identified as a protein with an apparent molecular mass of 45 kDa. Peptide sequences
were obtained from the protein and the corresponding cDNA was cloned. The function of the DiT1
protein and its substrate specificity were confirmed
by expression of the cDNA in yeast cells and functional reconstitution of the recombinant protein into
liposomes. Recent advances in the molecular cloning
of DiT2 and in the analysis of the in vivo function of
DiT1 by antisense repression in transgenic tobacco
plants will be discussed. In non-green tissues,
the reducing equivalents required for glutamate
formation by NADH-GOGAT are supplied by the
oxidative pentose phosphate pathway. Glucose
6-phosphate, the immediate precursor of the oxidative pentose phosphate pathway is generated in the
cytosol and imported into the plastids by the plastidic
glucose 6-phosphate/phosphate translocator.

Key words: Glucose 6-phosphateuphosphate translocator,

glutamateumalate translocator, glutamineuglutamate translocator, GSuGOGAT, photorespiration, 2-oxoglutarateumalate
translocator.

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Physiological characterization of plastidic

dicarboxylate translocators in C3-plants
Lehner and Heldt first reported on the characterization
of plastidic dicarboxylate transporters in spinach
chloroplasts (Lehner and Heldt, 1978). As demonstrated
by silicone-oil-filtration-centrifugation, these proteins
catalyse the uptake of 14C-labelled dicarboxylic acids
(e.g. 2-oxoglutarate, malate, succinate, aspartate, and
glutamate) in an antiport manner. The uptake showed
saturation kinetics and was stimulated by light. The
measurements of the Km and Ki values for the individual
dicarboxylates suggested the existence of more than one
translocator with overlapping substrate specificities. Woo
and Osmond analysed the ammonia and 2-oxoglutaratedependent O2 evolution in isolated chloroplasts and found
a strong stimulation of the (ammonia, 2-oxoglutarate)dependent O2 evolution by dicarboxylates (e.g. malate,
succinate and fumarate) (Woo and Osmond, 1982). The
calculated maximum rates of ammonia assimilation in
isolated chloroplasts were found to be sufficient to cope

with the reassimilation of ammonia generated in the

photorespiratory pathway in vivo. Using a reconstituted
spinach chloroplast system, Anderson and Osmond
found no significant effect of malate on the (glutamine,
2-oxoglutarate)-dependent O2 evolution, thereby supporting the suggestion that the observed stimulation
of (ammonia, 2-oxoglutarate)-dependent O2 evolution of
isolated chloroplasts by malate may be due to a promotion of 2-oxoglutarate uptake into chloroplasts by
malate (Anderson and Walker, 1983). However, Lehner
and Heldt had shown that the uptake of 2-oxoglutarate
is competitively inhibited by malate (Lehner and Heldt,
1978). Thus, the observed malate-stimulation of ammonia
assimilation by isolated chloroplasts would require an
entirely new mechanism for the transport of 2-oxoglutarate
across the chloroplast envelope membrane.
The two-translocator model for the transport
of dicarboxylic acids and glutamate in plastids

The mechanism of 2-oxoglutarate uptake into chloroplasts has been investigated further (Woo et al., 1987a;
Flugge et al., 1988) and it was shown that the chloroplast
envelope membrane contains at least two distinct
dicarboxylate transporters with partially overlapping substrate specificities, the 2-oxoglutarateumalate-translocator,
(DiT1) and the glutamateumalate-translocator (DiT2).
DiT1 was demonstrated to be specific for dicarboxylates (malate, succinate, fumarate, glutarate, and
2-oxoglutarate) whereas DiT2, in addition to the substrates transported by DiT1, also accepts the amino acids
glutamate and aspartate. A two-translocator model for
the transport of 2-oxoglutarate and glutamate was
proposed (Fig. 1A) that explained the stimulation of
2-oxoglutarate transport into isolated chloroplasts and the
stimulation of the (ammonia, 2-oxoglutarate)-dependent
oxygen evolution in isolated chloroplasts by malate
(Woo and Osmond, 1982). According to this model,
2-oxoglutarate is imported into the plastid in counterexchange with malate by DiT1 and is subsequently
converted to glutamate by GSuGOGAT. Glutamate is
then exported to the cytosol by DiT2, again in counterexchange with malate. In summary, this results in a
counter-exchange of 2-oxoglutarate with glutamate without a net malate transport. By inhibiting DiT2 with
high concentrations of glutamate, the kinetic properties
of DiT1 could be analysed in more detail (Yu and Woo,
1992a, b). The Km for the uptake of malate into isolated
spinach chloroplasts was found to be 2.7 mM and
0.6 mM for DiT1 and DiT2, respectively. The different
affinities of DiT1 and DiT2 for malate and the other
substrates provide the kinetic basis of the push and pull
mechanism for the transport of dicarboxylates that
was proposed by the two-translocator model. In potato
leaves, the concentration of malate at the end of the light

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photorespiration. Barley mutants defective in GS2

have been described previously (Blackwell et al., 1987;
Wallsgrove et al., 1987). These mutants developed
normally when grown under conditions that minimized
photorespiration. In air, however, they were unable to
grow. The studies using mutants of Arabidopsis and
barley conclusively demonstrated that both GS2 and
Fd-GOGAT are absolutely necessary for the reassimilation of ammonia released during photorespiration in
higher plants.
Fusion of the reporter gene GUS to the promoter
regions of the GS1 and GS2 genes from pea and subsequent transformation of the reporter constructs into
tobacco plants revealed a cell-specific expression pattern
of the GS2: :GUS fusion in photosynthetic cells, whereas
expression of the GS1: :GUS construct was restricted
to phloem cells. Immunocytochemical studies also
demonstrated that GS2 was localized to the plastids of
mesophyll cells whereas GS1 could be detected mainly in
the cytosol of phloem companion cells (Carvalho et al.,
1992). Since GS1 is also induced by wounding and during
senescence, differential roles for GS1 (synthesis of amino
acids for long-distance transport) and GS2 (synthesis of
amino acids for protein biosynthesis) have been suggested
(Masclaux et al., 2000).
Because the precursor for ammonia assimilation is synthesized in the cytosol anduor in the mitochondria (for
a recent review on the synthesis of 2-oxoglutarate, see
Lancien et al., 2000), plastids rely on a transport system
for the import of 2-oxoglutarate into the plastids and for
the export of the product of ammonia assimilation,
glutamate, from the plastids into the cytosol.

Plastidic transporters in C/N-metabolism

867

period was found to be 3.2 mM in the cytosol and

2.4 mM in the stroma. The concentrations of glutamate
in the cytosol and the stroma were determined to be
41 mM and 26.4 mM, respectively (Leidreiter et al.,
1995). Consequently, the export of glutamate from the
plastid stroma to the cytosol occurs against a concentration gradient and is driven by the inverse concentration
gradient for malate. For 2-oxoglutarate, no data on the
subcellular concentration are available. However, it can
be speculated that the concentration of 2-oxoglutarate
in the cytosol is in the low micromolar range and even
lower in the stroma (because GSuGOGAT represents a
strong sink for 2-oxoglutarate). Therefore, the import of
2-oxoglutarate is driven by a concentration gradient
towards the stroma and this, in turn, drives the export
of malate to the cytosol against a concentration gradient.
The question arises, why two transporters need to work
in concert. Considering the high concentration of glutamate in the cytosol in comparison with the low concentration of 2-oxoglutarate (at least three orders of
magnitude difference in concentration), it is obvious that
the transport of 2-oxoglutarate by one single transporter
with affinities for both glutamate and 2-oxoglutarate,
would always be out-competed by glutamate and no
transport of 2-oxoglutarate would occur. However, the
presence of two transporters with overlapping substrate specificities allows the import of 2-oxoglutarate
into the plastid stroma by DiT1 even in the presence of
high cytosolic concentrations of glutamate.
The plastidic glutamine translocator and
the three-translocator model

Using a double silicone layer filtering centrifugation

system, Yu and Woo characterized a specific glutamine

translocator from spinach and oat chloroplasts (Yu

and Woo, 1988). The transport of glutamine into the
chloroplast was greatly affected by the endogenous
dicarboxylate pools of plastids. In glutamine-preloaded
chloroplasts, the uptake of glutamine was competitively inhibited by glutamate only, but not by other
dicarboxylates that are substrates of DiT2, thus demonstrating that the glutamine transport is mediated by a
translocator different from DiT2. The combined action of
this putative glutamineuglutamate translocator and both
DiT1 and DiT2 opens the way to export glutamine in
exchange for 2-oxoglutarate, with no net glutamate and
malate transport, thereby providing the cytosol with
glutamine as a precursor, for example, for biosynthesis of
asparagine (Fig. 1B). The coupling of glutamine export
from plastids to the availability of glutamate in the
cytosol may act as a control mechanism circumventing
glutamine-depletion of the GSuGOGAT-cycle. In summary, it is likely that, in vivo, the transport of dicarboxylates, glutamate and glutamine occurs according to
the three-translocator model.

Ammonia assimilation and dicarboxylate

transport in gymnosperms
By contrast to the situation in C3-type angiosperms
(McNally et al., 1983), there is increasing evidence that
at least in some gymnosperm species GS1 is the dominant
isoform of GS and therefore most of the GS activity
is found in the cytosol and not in the plastid stroma
(Canton et al., 1993). An immunolocalization study of
GS1 showed that GS1 is localized not only in phloem
cells of pine seedlings, but also in mesophyll cells (GarcaGutierrez et al., 1998). A detailed analysis of GS1

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Fig. 1. The two-translocator model for transport of dicarboxylic acids and glutamate (A) and the three-translocator model for the transport
of dicarboxylates, glutamate and glutamine (B) into chloroplasts (1, DiT1; 2, DiT2; 3, glutamineuglutamate translocator). Cycling of substrates
between chloroplast stroma and the cytosol is indicated by broken lines whereas net fluxes are indicated by solid lines.

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Weber and Flugge

expression levels in Scots pine seedlings (Canton et al.,

1999) showed that GS1 mRNA levels increased upon
transfer of seedlings into the light. These findings
suggested that glutamine synthesis in pine seedlings by
glutamine synthetase mainly takes place in the cytosol
rather than in the plastid stroma. Since the glutamine
required for the de novo synthesis of glutamate by
plastidic Fd-GOGAT is supplied by cytosolic GS1, an
active glutamateuglutamine exchange able to cope with
the flux of ammonia from nitrate reduction and photorespiration has to be postulated. Two possible models for
the transport of 2-oxoglutarate, glutamate and glutamine
can be proposed.
Possible models for the transport of 2-oxoglutarate,
glutamate and glutamine into gymnosperm plastids

Role of the plastidic glucose

6-phosphate/phosphate translocator
during ammonia assimilation
Non-green plastids of heterotrophic tissues rely on
the provision of carbohydrates from the cytosol. The
imported carbon is used as a substrate for biosynthetic
pathways, for example, for the biosynthesis of starch or
fatty acids. Carbon can also be used to drive the oxidative
pentose phosphate pathway (OPPP), which is the major
source of reducing power required for the reduction of
nitrite and the biosynthesis of fatty acids and of amino
acids. The immediate substrate for the OPPP is glucose
6-phosphate (Glc6P) that is imported into the plastids
by the Glc6Puphosphate translocator (GPT; Kammerer

Fig. 2. Putative models for the transport processes in pine seedlings during ammonia assimilation by combined action of cytosolic GS1 and plastidic
Fd-GOGAT. (A) Two-translocator model involving DiT2 (2) and the glutamineuglutamate translocator (3). (B) Three-translocator model involving
DiT1 (1), DiT2 (2) and the glutamineuglutamate translocator (3). Cycling of substrates between chloroplast stroma and the cytosol is indicated by
broken lines whereas net fluxes are indicated by solid lines.

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One possible mode of transport could involve DiT2 and

the glutamine translocator working in concert as a double
translocator system for 2-oxoglutarate, glutamine and
glutamate (Fig. 2A). DiT2 would import 2-oxoglutarate
into the plastid stroma in counter-exchange with an
export of glutamate. Glutamate is converted to glutamine
in the cytosol by GS1 and imported into the plastid in
a counter-exchange for glutamate. In summary, this
would result in a net flux of 2-oxoglutarate to glutamate
whereas, in parallel, glutamine and glutamate would
cycle on DiT2 and the glutamine translocator, respectively. In this scenario, the activity of DiT1 would not
be required.
The other possible model would combine DiT1, DiT2
and the glutamine translocator to a three-translocator

model (Fig. 2B). In this model, glutamine formed by

GS1 in the cytosol is imported into the chloroplast in
a 1 : 1 counter-exchange with glutamate. The import of
2-oxoglutarate by DiT1 and the export of the second
molecule of glutamate resulting from Fd-GOGAT action
by DiT2 is coupled to malate counter-exchange in the
cascade-like push-and-pull mechanism already described
for the double translocator system.
In order to check which of the above-described models
describes best the in vivo situation, it will be necessary
to analyse the expression patterns of DiT1 and DiT2
in gymnosperms, such as pine seedlings, as well as the
transport characteristics of pine seedling chloroplasts. It
might be speculated that the three-translocator model
best describes the situation in vivo.

Plastidic transporters in C/N-metabolism

et al., 1998). The GPT thus links reactions located in

the cytosol, the conversion of sucrose to hexoses and
Glc6P, with the metabolism inside the plastid. Contrary
to photosynthetic tissues, the main source of ammonium
is not photorespiration but nitrate assimilation. Previous
studies have shown that nitrite reduction and the
subsequent synthesis of glutamate via GSuNAD(P)HGOGAT is strongly dependent on the provision of the
plastids with Glc6P and a functional OPPP that delivers
reductants for assimilatory processes in non-green
plastids (Bowsher et al., 1989, 1992; Fig. 3).

Purification and identification of the plastidic

2-oxoglutarate/malate translocator

Fig. 3. The role of the plastidic glucose 6-phosphateuphosphate translocator (GPT, 4) in supplying the plastidic oxidative pentose phosphate
pathway with the precursor glucose 6-phosphate.

cDNA cloning, expression in yeast cells and

functional characterization of DiT1
The identification of DiT1 as a component of the chloroplast envelope membrane (Menzlaff and Flugge, 1993)
was instrumental for cloning of the corresponding cDNA.
Weber et al. developed a preparative purification procedure for the 45 kDa protein based on its solubility in
chloroform (Weber et al., 1995). The protein could be
purified by preparative SDSPAGE in amounts sufficient
for subsequent cleavage with the endoproteinase Lys-C.
This approach yielded two peptide sequences of DiT1
that were used to synthesize corresponding oligonucleotides and subsequently to screen a spinach leaf cDNA
library.
A full-length cDNA was obtained, encoding a highly
hydrophobic protein of 569 amino acids with a predicted
molecular mass of 60.3 kDa and a calculated isoelectric
point of 9.6. A plastid targeting signal sequence of 90100
amino acid residues in length was predicted from the
protein sequence. The in vitro-translated protein was
found to be imported into intact isolated chloroplasts and
processed to its mature form.
By contrast to other known translocator proteins of
the plastid envelope membrane at that time (e.g. the triose
phosphateuphosphate translocator; Flugge et al., 1989)
that were found to form dimers of two identical subunits with six a-helices each, the DiT1 protein possesses
12 highly hydrophobic membrane-spanning segments.
Such a 12 transmembrane helix topology is found in
many bacterial transporters or transporters of the plasma
membrane. Later, a similar topology was also found for
other plastidic transporters, for example, the adenylate
translocator (Neuhaus et al., 1997) and the hexose translocator (Weber et al., 2000). Currently, it seems more
likely that the phosphate translocator family is rather
the exception than the rule in terms of its transmembrane
topology (for a recent review on plastid transporter
proteins see Neuhaus and Wagner, 2000).
Surprisingly, no significant homologies were found
to dicarboxylate transporter proteins from mitochondria
(Walker and Runswick, 1993; Taniguchi and Sugiyama,
1996) although both transporters share similar substrate
specificities and functions.
In order to confirm the identity of the DiT1 cDNA
with the proposed function, the cDNA was expressed in
yeast cells. Membrane proteins from yeast cells harbouring the transgene were isolated and reconstituted into
liposomes. The malate uptake into liposomes containing the recombinant protein was found to exceed the
transport activity of control liposomes by at least two
orders of magnitude. It could also be shown that the
recombinant protein possessed substrate specificities
that were identical to those of the authentic protein
(Weber et al., 1995).

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Menzlaff and Flugge reported on the identification of

the plastidic 2-oxoglutarateumalate translocator as a component of the plastid envelope membrane (Menzlaff and
Flugge, 1993). Detergent solubilized envelope membrane
proteins were separated by two successive column chromatography steps and the activity of DiT1 was monitored
by reconstitution of column fractions into liposomes
followed by transport measurements. DiT1 transport
activity could be assigned to a protein with an apparent
molecular mass of 45 kDa.
Purified, reconstituted DiT1 accepted malate, succinate, tartrate, glutarate, 2-oxoglutarate, and fumarate,
but not the amino acids aspartate and glutamate, as
counter-substrates for the exchange of malate. Thus, the
substrate specificity of purified, reconstituted DiT1 was in
accordance with the substrate specificity determined for
DiT1 in isolated spinach chloroplasts (Woo et al., 1987a, b;
Flugge et al., 1988) and therefore in accordance with the
function of DiT1 proposed in the two-translocator model
for the transport of dicarboxylates.

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Related sequences to DiT1 in plants

and bacteria
Related sequences in plants

A sequence homology search of the GenBank database

(Viridiplantae subsection) was performed to identify genes
that are homologous to DiT1 gene from spinach. These
homologues can be grouped into two related, but distinct
classes. The first group contains sequences from A.
thaliana, spinach, corn, and tobacco and is closely related

to spinach DiT1 (7085% identity at the protein level).

The second group (DiT2-group), containing sequences
from A. thaliana, Sorghum bicolor, Flaveria bidentis,
tobacco, and spinach shares only about 4555% identity
to the spinach DiT1 protein, but 7080% identical amino
acid residues with each other (see phylogenetic tree,
Fig. 4). The A. thaliana genome contains three genes
homologous to spinach DiT1, all located on chromosome 5. The DiT1 homologue is a single copy gene
whereas the two other genes fall into the DiT2-group

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Fig. 4. Unrooted phylogenetic tree of the dicarboxylate transporter family in plants and eubacteria. The first two letters of the acronyms indicate the
species (At, Arabidopsis thaliana; Bs, Bacillus subtilis; Cm, Chlamydia muridarum; Cp, Chlamydia pneumoniae; Ct, Chlamydia trachomatis; Ec,
Escherichia coli; Fb, Flaveria bidentis; Hi, Haemophilus influenzae; Nt, Nicotiana tabacum; Sa, Staphylococcus aureus; So, Spinacia oleracea; Zm, Zea
mays). DiT, dicarboxylate transporter; TST, tartrate succinate transporter; CST, citrate succinate transporter.

Plastidic transporters in C/N-metabolism

of sequences. The A. thaliana DiT2 genes are arranged

in tandem with a head to tail orientation. No other
significant homologies were found to other plant proteins.
A cDNA representing DiT2 from spinach has recently
been isolated and this cDNA was expressed in yeast.
Preliminary experiments showed that the reconstituted
recombinant DiT2 protein has a substrate specificity
comparable to DiT1 but, in addition to the substrates
transported by DiT1, accepts glutamate and aspartate as
counter-substrates for malate. It is therefore very likely
that the DiT2 group of genes encodes the general dicarboxylate translocator (glutamateumalate-translocator)
of the plastidic double-translocator system for dicarboxylates (P Renne, UI Flugge, A Weber, unpublished
results).
The E. coli citrate transporter CitT is related to DiT1

Proteins related to DiT1 in other bacterial species

Related proteins are also encoded by the genomes of

other bacteria like Heamophilus influenzae, Helicobacter
pylori, Staphylococcus aureus, Chlamydia trachomatis,
Chlamydia pneumoniae, and Bacillus subtilis. The protein
from B. subtilis shows the highest amino acid identity
(53%) followed by the proteins from C. trachomatis and

C. pneumoniae with about 48% amino acid identities.

DiT2 displays a somewhat lower identity with the
bacterial transporters, suggesting that DiT1 is more
closely related to the common ancestor of the bacterial
and plant transporters while DiT2 might have evolved
from DiT1 during the evolution of plants. The primary
function of DiT1 in early plant cells might have been
the indirect exchange of redox equivalents between the
endosymbiont and the host cell. For example, in
Chlamydia, 2-oxoglutarate is taken up from the host cell
by a DiT1 homologue in exchange for malate. The
oxidation of 2-oxoglutarate to malate leads to the
generation of reductants and of energy in the form of
GTP. Since no significant homologies are found to
proteins from cyanobacteria, it is tempting to speculate
that DiT1 might trace back to the genome of the host
cell of the endosymbiontic event that eventually led to
the evolution of a eukaryotic plant cell. No significant
homologies with proteins from fungi or animals were
found. Together with the bacterial transporters, DiT1 and
DiT2 form a class of plant and eubacterial transporters
involved in the transport of di- and tricarboxylic acids
(Fig. 4).

In vivo function of plastidic dicarboxylate

translocators
Somerville and Ogren isolated an EMS-mutagenized
photorespiratory mutant of A. thaliana (Somerville and
Ogren, 1983) that was shown to be defective in the
plastidic glutamateumalate-translocator (dct, Somerville
and Somerville, 1985). A corresponding mutant from
barley has also been described (Wallsgrove et al., 1986).
The A. thaliana dicarboxylate translocator mutant dct

The A. thaliana mutant was shown to be not viable under

ambient CO2-conditions, but grew like the wild type
under elevated CO2 concentrations that suppress photorespiration. The phenotype was attributed to the inability
of the mutant to carry out a functional photorespiratory
pathway. A defect in a plastidic dicarboxylate translocator would lead to a deficiency in the reassimilation of
ammonia resulting from glycine-decarboxylase activity
either because of a lack in the supply of the GSuGOGAT
cycle with 2-oxoglutarate, the precursor for ammonia
assimilation, or a decreased export capacity for glutamate
(or both). In addition, either case would lead to a shortage in glutamate supply for the formation of glycine from
glyoxylate in the peroxisomes, leading to a build-up
of toxic photorespiratory intermediates in the mutant.
Furthermore, a block in photorespiratory cycling
causes a depletion of Calvin cycle intermediates. From
the physiological characterization, it is difficult to decide
whether the mutant is deficient in the activity of either

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Most Escherichia coli strains are unable to grow on citrate

under oxic conditions. Under anaerobic conditions, however, E. coli is able to grow on citrate in the presence of an
oxidizable co-substrate (e.g. glucose or glycerol). Citrate
is taken up into the cell and split into acetate and
oxaloacetate by citrate lyase (Lutgens and Gottschalk,
1980). Oxaloacetate is then reduced to succinate by concerted reactions of malate dehydrogenase, fumarase and
fumarate reductase. The required reducing equivalents
are generated by the oxidation of the co-substrate
(e.g. glucose or glycerol). Two enzymes are specifically
required for the fermentation of the tricarboxylic acid, a
citrate uptake system and citrate lyase. Pos et al. reported
on an open reading frame (designated CitT ) located at
13.90 min on the E. coli chromosome between the RNA
(RNAse I) and the citrate lyase genes that encodes a
citrate carrier (Pos et al., 1998). E. coli cells transformed
with a plasmid expressing CitT were able to grow on
citrate under aerobic conditions. It could be also shown
that CitT catalyses either an homologous exchange of citrate or a heterologous exchange with succinate, fumarate,
or tartrate (Pos et al., 1998). Since succinate is the endproduct of citrate fermentation in E.coli, it is likely that
CitT functions in vivo as a citrateusuccinate antiporter.
CitT is a highly hydrophobic protein (487 amino acids,
53.1 kDa) with 12 putative transmembrane helices. It is
related to the 2-oxoglutarateumalate translocator from
spinach chloroplasts (SoDiT) with the amino acid identity
to DiT1 from spinach being about 34%.

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insertional mutants in DiT1 and DiT2.1 is currently

underway.
Antisense repression of DiT1 in transgenic
tobacco plants

Fig. 5. Phenotype of a transgenic tobacco line showing antisense

repression of DiT1.

DiT1 or DiT2. Since the complete sequence of the

Arabidopsis genome is now available, it should be possible
to search for mutations in the three genes related to
spinach DiT1 in the Arabidopsis genome in order to
identify the gene defective in dct.

Wallsgrove et al. isolated a barley mutant (RPr 79u2) that

is defective in chloroplast dicarboxylate transport
(Wallsgrove et al., 1986). This mutant resembles in many
aspects the Arabidopsis mutant dct, including the labelling
pattern of serine, glycine, glycerate, and malate following
fixation of 14CO2, the rapid rise in glutamine content and
the fall in glutamate content in leaves upon transfer from
non-photorespiratory conditions to air. In contrast to
dct, the barley mutant does not die in ambient air unless
the temperature and irradiance are high. The survival
of RPr 79u2 in ambient air might be due to the build-up of
2-oxoglutarate levels (up to 10-fold in comparison to the
wild type) that might overcome the deficiency in chloroplast dicarboxylate transport (probably in DiT1) by
allowing DiT2 to operate as a 2-oxoglutarateuglutamate
exchanger under these conditions.
Knockout lines for plastidic dicarboxylate translocators

The availability of large collections of T-DNA tagged

A. thaliana insertion mutants (Krysan et al., 1999;
Sussman et al., 2000) enabled a PCR-based reverse
genetic screen to be set up to identify mutants that are
defective in either of the three DiT-related genes. A
homozygous knockout line for A. thaliana DiT2.2 was
Kolukisaoglu, B Schulz, A Weber, unpubisolated (U
lished results) but, surprisingly, the mutant does not
display an obvious phenotype. This might be due to a
redundancy in gene function with DiT2.1 or to a tissue
or cell specific expression pattern of the DiT2.2 gene
that does not lead to a visible phenotype in the aerial
parts of the plant. As judged from the availability of
A. thaliana ESTs for DiT2.1 and DiT2.2, it can be
speculated that DiT2.1 is expressed at higher levels and
more ubiquitous as compared to DiT2.2. A search for

Acknowledgements
We thank Peter Westhoff and Uta Dressen (University of
Dusseldorf, Germany) for supplying us with the cDNA
sequences of DiT2 from Flaveria bidentis and Sorghum bicolor.

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