Journal of Experimental Botany, Vol. 58, No. 5, pp. 1109–1118, 2007
doi:10.1093/jxb/erl269 Advance Access publication 13 January, 2007
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
The effect of Glc6P uptake and its subsequent oxidation
within pea root plastids on nitrite reduction and
glutamate synthesis
Caroline G. Bowsher1,*, Anne E. Lacey1, Guy T. Hanke2, David T. Clarkson3, Les R. Saker3,
Ineke Stulen4 and Michael J. Emes5
1
Faculty of Life Sciences, The University of Manchester, 3.614 Stopford Building, Oxford Road,
Manchester M13 9PT, UK
2
Institute for Protein Research, Osaka University, Osaka 565 0871, Japan
3
4
5
University of Bristol, Long Ashton Research Station, Bristol BS41 9AF, UK
University of Groningen, Laboratory of Plant Physiology, PO Box 14, 97500 AA, Haren, The Netherlands
College of Biological Science, University of Guelph, Guelph N1G 2W1, Canada
Received 25 July 2006; Revised 14 November 2006; Accepted 16 November 2006
Abstract
In roots, nitrate assimilation is dependent upon a supply of reductant that is initially generated by oxidative
metabolism including the pentose phosphate pathway
(OPPP). The uptake of nitrite into the plastids and its
subsequent reduction by nitrite reductase (NiR) and
glutamate synthase (GOGAT) are potentially important
control points that may affect nitrate assimilation. To
support the operation of the OPPP there is a need for
glucose 6-phosphate (Glc6P) to be imported into the
plastids by the glucose phosphate translocator (GPT).
Competitive inhibitors of Glc6P uptake had little impact
on the rate of Glc6P-dependent nitrite reduction. Nitrite
uptake into plastids, using 13N labelled nitrite, was
shown to be by passive diffusion. Flux through the
OPPP during nitrite reduction and glutamate synthesis
in purified plastids was followed by monitoring the
release of 14CO2 from [1-14C]-Glc6P. The results suggest that the flux through the OPPP is maximal when
NiR operates at maximal capacity and could not
respond further to the increased demand for reductant
caused by the concurrent operation of NiR and
GOGAT. Simultaneous nitrite reduction and glutamate
synthesis resulted in decreased rates of both enzy-
matic reactions. The enzyme activity of glucose 6phosphate dehydrogenase (G6PDH), the enzyme supporting the first step of the OPPP, was induced by
external nitrate supply. The maximum catalytic activity
of G6PDH was determined to be more than sufficient to
support the reductant requirements of both NiR and
GOGAT. These data are discussed in terms of competition between NiR and GOGAT for the provision of
reductant generated by the OPPP.
Key words: Glucose-6-phosphate dehydrogenase, glucose6-phosphate uptake, glutamate synthase, nitrite uptake, nitrite
reductase maximum catalytic activity, oxidative pentose
phosphate pathway competition.
Introduction
In higher plants, nitrogen assimilation involves the
enzymes nitrate reductase (NR, EC 1.6.6.2), nitrite reductase (NiR, 1.7.7.1), glutamine synthetase (GS, 6.1.1.3),
and glutamate synthase (GOGAT, EC 1.4.1.13 and
1.4.7.1; Crawford, 1995; Lam et al., 1996). There is some
divergence in higher plants between those which are
* To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: DHAP, dihydroxyacetone phosphate; DIDS, 4, 4#-diisothiocyanatostilbene-2-2#-disulphonic acid; G6PDH, glucose-6-phosphate
dehydrogenase; Glc6P, glucose-6-phosphate; Gln, glutamine; Glu, glutamate; GOGAT, glutamate synthase; GS, glutamine synthetase; GPT, Glc6P/
phosphate translocator; HP, hexose phosphates; NiR, nitrite reductase; OPPP, oxidative pentose phosphate pathway; PEP, phosphoenolpyruvate; PPT,
phosphoenolpyruvate/phosphate translocator; Pi, inorganic phosphate; PGA, phosphoglyceric acid; TPT, triose phosphate/phosphate translocator.
ª 2007 The Author(s).
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1110 Bowsher et al.
considered to be predominantly leaf assimilators, and those
which assimilate nitrate mainly in the root such as legumes
(Andrews, 1986; Pate, 1980). Nitrate assimilation is a highly
compartmentalized system as NR is located in the cytosol,
whilst NiR, a proportion of the GS, and all the GOGAT are
found in the plastids. The available data from studies on
transgenic and genetically manipulated plants indicates that
the levels of these enzymes do not limit primary nitrate
assimilation and therefore crop yield (Andrews et al., 2004;
Good et al., 2004). The process in plastids is also
dependent upon a supply of reducing power. Reductant is
generated photosynthetically in leaf tissue. Clearly, in nonphotosynthetic tissue this is not the case and, indeed, the
generation of reducing power by oxidative metabolism
could also have an impact on nitrogen assimilation.
There is good evidence that, in root plastids, the pentose
phosphate pathway generates reductant in the form of
NADPH, which is used subsequently to drive nitrogen
assimilatory reactions (Bowsher et al., 1989, 1992). The
oxidative reactions of the pentose phosphate pathway
(OPPP) are capable of operating in both the plastids and
the cytosol (Debnam and Emes, 2000; Schnarrenberger
et al., 1995). Whilst there appear to be species differences
in the localization of the non-oxidative reactions in the
cytosol, the complete pathway has been found in plastids
of all species examined thus far. It is the initial conversion
of glucose 6-phosphate (Glc6P) to ribose 5-phosphate,
catalysed by glucose 6-phosphate dehydrogenase
(G6PDH, EC 1.1.1.49) and 6 phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44), which produces two
molecules of NADPH. Six members of the G6PDH gene
family have been identified in Arabidopsis thaliana, based
on transit peptide analysis it is predicted that there are two
cytosolic and four plastidic isoforms (Kruger and von
Schaewen, 2003; Wakao and Benning, 2005). Five
members of the 6PGDH gene family have also been
identified of which at least two of these are thought to be
plastidic in location (Kruger and von Schaewen, 2003).
Based on the expression of genes encoding G6PDH and
6PGDH from A. thaliana following transfer to nitratecontaining medium, their involvement in providing
NADPH for ferredoxin-dependent reactions has been implicated (Wang et al., 2000). A 12–27-fold increase in
both enzymes activities in maize root plastids after 24 h
treatment with 10 mM nitrate has also been reported
(Redinbaugh and Campbell, 1998). Furthermore, increase
in the expression of the P2 form of G6PDH from roots of
Nicotiana tabacum (Knight et al., 2001) and Lycoperscion
esculentum (Wang et al., 2001) in response to nitrate and
from Hordeum vulgare root plastids in response to
ammonium/glutamate (Esposito et al., 2001b) have also
been reported.
The ability to monitor the flux through the OPPP using
purified root plastids has allowed the demonstration of
increased carbohydrate oxidation in response to nitrite
reduction (Bowsher et al., 1989) and glutamate synthesis
(Bowsher et al., 1992; Esposito et al., 2003). This
approach has also provided information on the stoichiometry of the interaction between pathways of carbon and
nitrogen metabolism (Hartwell et al., 1996). Furthermore
the P2 form of G6PDH has been confirmed as supporting
the increased flux through the OPPP (Esposito et al.,
2003). Finally, when Glc6P is added alone or simultaneously with nitrite to barley root plastids, the increased
NADPH/NADP ratio observed is consistent with the
activation of G6PDH enzyme activity and the OPPP
(Wright et al., 1997). The reactions catalysed by NiR and
GOGAT both require reduced ferredoxin as a substrate.
The OPPP-generated NADPH acts as the initial reductant
to generate reduced ferredoxin via a root-specific ferredoxin-NADP oxidoreductase (FNR, EC 1.18.12). FNR
transcript and enzyme activity is co-induced with rootspecific ferredoxin during the induction of nitrate assimilation (Bowsher et al., 1993; Matsumura et al., 1997;
Ritchie et al., 1994).
Using pea root plastids, it has previously been shown
that Glc6P can be taken up and supports nitrite reduction
and the biosynthesis of glutamate (Bowsher et al., 1989,
1992). In contrast to photosynthetic tissues, there is a need
to import carbon into the plastids of non-photosynthetic
tissue as a source of energy to drive biosynthetic reactions. Depending on the tissue examined and the plastid
type, a range of phosphate translocators has been identified which are capable of importing carbon precursors.
The triose phosphate/phosphate translocator (TPT) mediates the export of fixed carbon in the form of triose
phosphates and 3-phosphoglycerate in exchange for inorganic phosphate (Fliege et al., 1978). Although it is not
clear whether the TPT is expressed in roots (Flugge, 1999;
Knight and Gray, 1994), more recently, two novel plastid
transporters have been identified which show higher expression in roots. The plastidic PEP/phosphate translocator (PPT), which transports phosphoenolpyruvate
(PEP) in exchange with inorganic phosphate (Fischer
et al., 1997), and the glucose-6-phosphate/phosphate
translocator (GPT), which counter exchanges Glc6P with
inorganic phosphate and triose phosphates. This latter
carrier is analogous to the chloroplast TPT, but has
a much higher affinity for Glc6P (Borchert et al., 1989,
Flugge, 1999; Kammerer et al., 1998). At the protein
level, the GPT has low but significant similarity to the
TPT (Flugge, 1999) and the PPT (Fischer et al., 1997).
The proposed physiological function of the GPT is to
facilitate the import of Glc6P for use as an OPPP substrate. In the Arabidopsis genome two paralogous GPT
genes have been identified, AtGPT1 and AtGPT2 (Knappe
et al., 2003). Although both GPTs appear functional in
planta, the ubiquitously expressed AtGPT1, like pea GPT,
is expressed in roots and is thought to be the major GPT
involved in Glc6P transport into heterotrophic plastids.
Glc6P uptake and oxidation in pea root plastids 1111
Loss of AtGPT1 function disrupts the OPPP and has been
proposed to affect the reducing power supply needed for
fatty acid biosynthesis, leading to less lipid body
formation and disintegration of membrane systems, resulting in impaired pollen maturation and embryo sac
development (Niewiadomski et al., 2005). Glc6P import
into plastids potentially impacts not only the capacity of
the plastids to sustain the OPPP, but also other related
metabolic processes including nitrate assimilation via NiR
and GOGAT.
The reduction of nitrite and synthesis of glutamate are
dependent upon both the uptake of Glc6P and its subsequent oxidation within the organelle. In this paper, the
relative importance of these two aspects in controlling
nitrogen assimilation using purified root plastids of Pisum
sativum is investigated. The approaches taken in answering these questions were (i) to determine the effect of
inhibition of Glc6P uptake on nitrite reduction; (ii) to investigate the mechanism of nitrite entry into the organelle;
and (iii) to determine whether there is competition for
reductant between the processes of nitrite reduction and
glutamate synthesis. The results demonstrate that there is
competition for electrons between nitrite reductase and
glutamate synthase and that the OPPP is not able to
produce sufficient reductant to support the optimal activities of these two enzymes simultaneously. This suggests that the oxidation of Glc6P through the OPPP may
place a significant constraint on the reactions of nitrogen
assimilation in plastids.
Materials and methods
Chemicals
All reagents were of ‘AR’ grade where possible. Cofactors,
enzymes and substrates were purchased from BDH, Poole, Dorset
(UK), Boehringer Mannheim (FRG), or Sigma, Poole, Dorset,
(UK). Percoll was purchased from Amersham Pharmacia, Milton
Keynes (UK). [1-14C]-glucose-6-phosphate was from New England
Nuclear, Southampton (UK) and Silicone AR200 oil was from
Water Chemicals UK Ltd.
Plant material
Pea (Pisum sativum L. cv. Early onward) seeds purchased from
Yates Seed Breeders and Merchants (Macclesfield, Cheshire UK),
were germinated in the dark for 5 d at 25 C as described previously
(Emes and England, 1986). On day 5, 24 h prior to extraction, the
peas were watered three times with 50 mM KNO3.
Extraction and purification of root plastids
Plastids were prepared from 150–300 g of roots, based on the
method described previously (Bowsher et al., 1989). Intact plastids
were maintained in buffer A containing 50 mM N-[2-hydroxy-1,1bis(hydroxymethyl)ethyl]glycine (Tricine-NaOH, pH 7.9, containing 330 mM sorbitol, 1 mM ethyldiaminetetra-acetic acid (EDTA),
1 mM MgCl2). When required, ‘broken’ plastids were ruptured
osmotically by resuspending in distilled water, followed by the
addition of an equal volume of double-strength buffer A to
standardize the salt concentration. In some instances Triton X-100
was added to a final concentration of 0.1% (v/v), although this had
no effect on the results obtained.
Glucose-6-phosphate-dependent nitrite reductase/glutamate
synthase activity
The assay contained 150 mM 2-amino-2-(hydroxymethyl)-1,3propanediol (Tris)-HCl, pH 8.0, 0–10 mM glucose-6-phosphate,
substrate (1 mM nitrite, for nitrite reductase and/or 5 mM glutamine
and 5 mM 2-oxoglutarate for glutamate synthase) and intact or
broken plastids (550 lg protein). All solutions were osmotically
buffered with 330 mM sorbitol. At known times the reaction was
stopped by the addition of either 0.5% (v/v) acetic acid or 0.4 mM
5-sulphosalicylic acid, respectively. Samples were removed for
nitrite determination or glutamate estimation as described previously (Bowsher et al., 1989, 1992). All assays were corrected for
nitrite reduction or glutamate synthesis in broken plastids by
carrying out the same experiments using organelles osmotically
lysed and subtracting these values from those obtained with intact
preparations.
Glucose-6-phosphate dehydrogenase
G6PDH was assayed based on the method of Fowler and ap Rees
(1970).
Protein determination
Protein was measured using a commercial Bio-Rad (Watford, UK)
protein method based on Bradford (1976) with thyroglobulin as the
protein standard.
Measurement of 14CO2 evolution from [1-14C]-glucose-6phosphate
All assays were carried out in a final volume of 1 cm3 in the
presence of nitrite and/or glutamine and 2-oxoglutarate as previously described (Bowsher et al., 1989, 1992).
Transport measurements
Silicone oil centrifugation was carried out based on the method of
Emes and Traska (1987). All reagents were made up in 330 mM
sorbitol. For Glc6P uptake measurements, 90 ll root plastids
(approximately 100 lg protein, resuspended in buffer A) were
incubated for 15 min at room temperature with 10 ll 330 mM
sorbitol, or 10 ll 330 mM sorbitol containing DIDS, phosphoglyceric acid (PGA), inorganic orthophosphate (Pi), or dihydroxyacetone phosphate (DHAP). At the end of this period, U-[14C]-Glc6P
(specific activity¼3.7 kBq lmol1) was added to the mix for 30 s at
20 C. Substrate uptake was stopped by centrifugation at 13 000 g
for 120 s through 50 ll silicone oil (AR200). Pelleted material was
removed by excising the bottom of the tube. The lower portion of the
cut tube was placed in Ecoscint scintillation fluid and thoroughly
shaken before radioisotope counting. Residual silicone oil did not
cause an increase in quenching. The volume of organelles sedimented
was calculated from measurements with tritiated water and corrected
for the organelle-free space with 14C-dextran (Heldt, 1980). This
latter value varied between 2% and 6% of the space occupied by
3
H2O in the sediment. Estimation of the ‘sorbitol-permeable’ space
with U-14C sorbitol gave values which were not significantly
different from those obtained with 14C-dextran.
To determine nitrite uptake a 6.5 ml aliquot of 13N-labelled
nitrate (prepared by proton irradiation of pure water at the Positron
Emission Tomography Centre, University Hospital, Groningen, The
1112 Bowsher et al.
Results
It has previously been demonstrated that exogenous
glucose-6-phosphate (Glc6P) is capable of supporting
nitrite reduction and glutamate formation in pea root
plastids (Bowsher et al., 1989, 1992; Emes and Fowler,
1983). The importance of the Glc6P/phosphate translocator (GTP) in controlling the Glc6P-dependent nitrite
reduction was examined in purified plastids by using
substrates which are competitive biochemical inhibitors of
the transporter (Figs 1, 2). As would be expected of
a protein with phosphate binding activity, DIDS was
nmoles nitrite
reduced.mg protein-1
1000
800
600
400
200
0
0
10
20
30
40
50
DIDS Concentration (mM)
Fig. 1. The effect of DIDS on Glc6P-dependent nitrite reduction. Intact
pea root plastids were incubated with either 0.5 mM (filled diamonds)
or 5 mM (filled squares) Glc6P, 1 mM NO
2 and varying concentration
of DIDS. Each value represents the mean 6SEM from at least three
independent measurements.
120
% Glc6P uptake
Netherlands) was first converted to 13N-nitrite based on the method
developed by Brewer et al. (1965) in which nitrate is reduced in the
presence of cadmium and EDTA (see Grasshoff, 1964; Wood et al.,
1967, for a discussion of the chemistry). One ml of nitrate/EDTA
solution (2.80g Na2-EDTA, 246 ll 1000 ppm KNO3, adjusted to
pH 6.8 with KOH and made up to 25 ml) was pipetted into a 10 ml
glass vial in a lead pot. A 6.5 ml aliquot of 13N-labelled nitrate, that
also contained some 13NO
2 (prepared by proton irradiation of pure
water at the Positron Emission Tomography Centre, University
Hospital, Groningen, The Netherlands), was withdrawn via a hypodermic needle from the sealed multidose glass bottle, located within
a lead pot, into a 25 ml syringe, which itself was housed in a lead
jacket with lead glass window and added, with mixing, to the vial to
give a solution of 100 lmol KNO3 in 1.5% (w/v) EDTA.
13
The solution containing 13NO
NO
3,
2 , unlabelled NO3 , and
1
EDTA; was pumped by a peristaltic pump at 0.32 ml min through
a cadmium reduction coil. The coil consisted of an 80 cm length of
polythene tubing, internal diameter 2.45 mm (Portex Ltd., Hythe,
Kent, size 800/500/540) filled with cadmium filings (produced by
rasp from stick cadmium metal and gently sieved to remove the
smaller particles that would pass through a size 35 mesh). The coil
was lightly plugged at each end with silica wool and kept filled with
either water or the solution from the previous run to prevent
oxidation and to maintain chemical reactivity. At the start of the
reduction process the water or previous solution entrapped in the
coil was flushed to waste by passing the fresh 13N-labelled mixture
through for about 7 min (until the outflow was strongly radioactive).
A resin cation exchange column (1 ml zeo-karb 225, in a 1 ml
disposable syringe body) was then rinsed for 1–2 min by the
following aliquot of solution. The subsequent 2–2.5 ml of the
solution was collected in a polythene scintillation vial insert in
a double lead transport container, in which it was conveyed to the
experimental location. The final 2–3 ml of the treated solution was
collected and frozen for later analysis of nitrite and cadmium
concentrations.
Samples were retained for analysis of nitrite and stored frozen for
1–2 d. After thawing, a 0.25 ml aliquot of the sample was diluted to
1 ml with water and nitrite determined using diazo-dye coupling
reagents as described previously (Bowsher et al., 1989).
For the determination of cadmium content, the sample was
diluted 1:100 or 1:50 and the atomic absorption was measured at
228.8 nm (on a Fisons Atomic Absorption Spectrophotometer, at
the Institute of Soil Fertility, Groningen) and compared with a 0–3
lg Cd2+ ml1 Standard (0.267 lmol).
The uptake of 13N-nitrite into plastids was then determined by
incubating root plastids with 0–125 lM 13N-labelled nitrite for 30 s
at 20 C and subsequent determinations made as described above,
accounting for the number of half-lives of 13N lost during the
procedure.
100
80
60
40
20
0
0
1
2
3
4
5
Inhibitor Concentration (mM)
Fig. 2. Inhibition of the uptake of Glc6P into pea root plastids by
3PGA (filled diamonds), DHAP (filled triangles), or Pi (filled squares).
The plastids were preincubated with 0–5 mM inhibitor The uptake of
0.1 mM [U-14C]Glc6P was stopped after 30 s by silicone oil-filtering
centrifugation and the amount of [U-14C]-Glc6P taken up was determined. The values are the means from at least three independent
measurements.
a strong inhibitor of Glc6P uptake, and hence nitrite
reduction (Fig. 1). Because the effect of DIDS is timedependent and involves covalent modification of the target
protein, it was not possible to compare directly the effects
of DIDS on Glc6P uptake (which is measured over
seconds) with its effect on Glc6P-dependent nitrite reduction (measured over tens of minutes). Consequently, the
choice was made to use known physiological competitive
inhibitors of Glc6P uptake in order to determine their
relative impact on nitrite reduction and Glc6P uptake.
Figure 2 shows that Glc6P transport is inhibited by PGA,
Pi, and dihydroxyacetone phosphate, producing approximately 50% inhibition at the highest concentrations used.
By contrast, none of these compounds had any significant
effect on the rate of Glc6P-dependent nitrite reduction,
even at concentrations up to 20 mM (Fig. 3a, b, c). The
implication of these results is that a substantial decrease in
the capacity for Glc6P uptake has little impact on Glc6Pcoupled nitrite reduction which is being measured within
the organelle. It would seem, therefore, that the GPT exerts
little overall control over the process of nitrite reduction.
Likewise there was no effect by any of these compounds
Glc6P uptake and oxidation in pea root plastids 1113
1200
1000
1000
800
800
600
600
400
400
200
200
0
1
0
2
3
4
5
b
1000
800
600
400
200
0
1
0
2
3
4
5
c
1000
800
nmoles 14CO2 evolved.h-1. mg protein-1
nmoles nitrite reduced. h-1. mg protein-1
d
a
1200
0
0
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
e
1400
1200
1000
800
600
400
200
0
0
f
1200
1000
800
600
600
400
400
200
200
0
0
0
1
2
3
4
0
5
Glc6P Concentration (mM)
Glc6P Concentration (mM)
on 14CO2 evolution from [l-14C]-Glc6P (diagnostic of the
flux through the OPPP; Bowsher et al., 1989) supporting
the view that the activity of the OPPP was unimpaired by
the decrease in the capacity for Glc6P uptake which these
competitive inhibitors would produce (Fig. 3 d, e, f).
A second site of possible regulation of Glc6P-dependent
nitrite reduction is the movement of nitrite across the
plastid envelope. Uptake of NO
2 , however, appeared to
be linear with concentration and did not saturate at
concentrations likely to be encountered in vivo even in
excess of 100 lM (Fig. 4). The data suggest that NO
2
crosses the plastid envelope by passive diffusion. Certainly, there was no direct evidence for a carrier-mediated
process and uptake was not impaired in the presence of
mM concentrations of other anions such as Pi and SO2
4 ,
nor by DIDS (data not shown). Furthermore, when 13NO
2
uptake was measured in the presence of 10 mM Glc6P
a more than 3-fold increase in the rate of diffusion of
nitrite into the plastids was recorded (data not shown).
This suggests that an increase in the availability of Glc6P
to support reduction was increasing the diffusion gradient
of NO
2 across the plastid envelope.
The above results suggest that the process of nitrite
reduction is most likely limited by events within the
organelle, rather than by the entry of substrates. Using
Uptake of NO2- into plastids
(nmol NO2-. µl plastid)
Fig. 3. The effect of the metabolites 3PGA (a, d), DHAP (b, e), and Pi (c, f) on Glc6P-dependent nitrite reduction (a, b, c) and the release of 14CO2
(d, e, f) from [1-14C]-Glc6P. Intact plastids were supplied with 0 (filled diamonds), 5 mM (filled squares), or 20 mM (filled triangles) metabolite in
the presence of varying concentrations of [1-14C] Glc6P and 1 mM NO
2 . The values are the means from at least three independent measurements.
0.6
0.5
0.4
0.3
0.2
0.1
0
0
50
100
150
Concentration of NO2- (µM)
Fig. 4. Nitrite uptake by pea root plastids. The uptake of 13NO
2 was
stopped after 30 s by silicone oil-filtering centrifugation and the amount
of 13NO
2 taken up was determined. The experiment was repeated at
least three times. Representative data from one independent experiment
is shown.
isotopically labelled Glc6P it is possible to investigate the
effect of nitrite reduction and/or glutamate synthesis on
carbohydrate oxidation by measuring the release of
14
CO2. Previously, it has been shown that nitrite reduction
and glutamate synthesis in root plastids are directly
responsible for an increase in CO2 evolution specifically
and only from C atom 1 of Glc6P, implying an increased
flux of C through the OPPP (Bowsher et al., 1989, 1992).
The relationship between flux through the OPPP, as
1114 Bowsher et al.
nmoles 14CO2 evolved.
h-1. mg protein-1
a
1600
1400
1200
1000
800
600
400
200
0
nmoles nitrite reduced.
h-1. mg protein-1
0
2
4
6
8
10
2
4
6
8
10
4
6
8
10
b
1200
1000
800
600
400
200
0
0
nmoles glutamate formed.
h-1. mg protein-1
measured by 14CO2 evolution from [1-14C]-Glc6P (Fig. 5a),
and the ability of the plastid preparations to support reductive assimilation and biosynthesis measured as nitrite
reduction (Fig. 5b) and glutamate formation (Fig. 5c) were
therefore examined. Figure 5a demonstrates that the
evolution of 1-14CO2 increases with Glc6P concentration
in the presence of the NiR substrate, nitrite, or in the
presence of the GOGAT substrates, glutamine and 2oxoglutarate. Both the NiR and GOGAT reactions are
dependent on a supply of reduced ferredoxin which is
initiated by the generation of NADPH through the OPPP.
The response to varied Glc6P presumably reflects, at least
in part, the kinetics of G6PDH and the other enzymes of
the OPPP in utilizing the substrate and products formed as
has been observed previously (Bowsher et al., 1989).
Evolution of 1-14CO2 was less in the presence of glutamine and 2-oxoglutarate when compared with nitrite,
presumably reflecting the relative maximal catalytic
activities of NiR and GOGAT. The reduction of 1 mol of
nitrite requires six electrons, whereas the conversion of
glutamine and 2-oxoglutarate to 2 mol of glutamate
requires only two electrons. To investigate whether there
is competition between NiR and GOGAT for reductant
when operating simultaneously, the impact of both
reactions on flux through the OPPP was examined. When
plastids were supplied with saturating substrate concentrations for NiR, (nitrite), and GOGAT, (glutamine and
2-oxoglutarate), then at all concentrations of Glc6P, the
amount of CO2 released was only equivalent to the rate
seen when nitrite reduction alone was occurring (Fig. 5a).
These results suggest that at any given operating concentration of Glc6P, irrespective of the demand for reductant,
the flux through the OPPP cannot be increased above that
which is generated when supporting nitrite reduction
alone, even when there is an additional ‘sink’ for electrons
effected by the substrates for GOGAT. Experiments were
therefore undertaken to measure the impact glutamate
synthesis has on Glc6P-dependent nitrite reduction and
vice versa. In Fig. 5b it can be seen that the rate of nitrite
reduction is decreased in the presence of the substrates for
the GOGAT reaction, glutamine and 2-oxoglutarate. This
was true at all concentrations of Glc6P, including substrate levels well in excess of the Km for Glc6P uptake or
G6PDH. Similarly, at all concentrations of Glc6P, the
formation of glutamate was reduced by incubating plastids
with the substrate for nitrite reduction in addition to
glutamine and 2-oxoglutarate (Fig. 5c).
It has previously been shown that the increase in
GOGAT activity that occurs through supplying plastids
with an increasing glutamine concentration in the presence
of a fixed amount of 2-oxoglutarate, leads to an increased
flux of carbon through the OPPP (Bowsher et al., 1992).
This method of titrating in GOGAT activity by increasing
the availability of glutamine substrate was exploited to
examine the competition between NiR and GOGAT for
c
1600
1400
1200
1000
800
600
400
200
0
0
2
Glc6P Concentration (mM)
Fig. 5. The effect of different concentrations of [1-14C]-Glc6P on (a)
evolution of 14CO2, (b) nitrite reduction, and (c) glutamate formation.
Pea root plastids incubated with different concentrations of [1-14C]Glc6P and either 1 mM sodium nitrite (filled diamonds) or 5 mM Gln+5
mM 2-oxoglutarate (filled squares) or 1 mM sodium nitrite, 5 mM
Gln+5 mM 2-oxoglutarate (filled triangles). Each value represents the
mean 6SEM from at least three independent experiments.
reducing power generated by the OPPP (Fig. 6). Intact
plastids were supplied with 1 mM nitrite and nitrite
reduction and the OPPP flux were followed at high
(5 mM) and low (0.5 mM) concentrations of Glc6P. The
activity of GOGAT increased with the concentration of
glutamine against a fixed amount of 2-oxoglutarate at both
Glc6P concentrations (Fig. 6a). Figure 6b shows that the
evolution of 14CO2 from 1-14C-Glc6P, and hence the
OPPP flux, was unaffected by increasing the glutamine
level. By contrast, it was clear that there was a steady
decrease in nitrite reduction as the glutamine concentration
was raised at either Glc6P concentration used (Fig. 6c). In
effect, the GOGAT reaction was being titrated against
nitrite reduction in a competition for reducing power.
The results of these experiments showed that there was
competition for electrons between nitrite reductase and
nmoles 14CO2 evolved.
h-1. mg protein-1
nmoles glutamate formed.
h-1. mg protein-1
Glc6P uptake and oxidation in pea root plastids 1115
mg protein h1 between 10–25 mM external nitrate.
The co-ordinated induction of G6PDH activity is clearly
a response to the increased demand for reducing power as
a result of the induction of nitrate assimilation.
a
800
700
600
500
400
300
200
100
Discussion
0
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
b
1200
1000
800
600
400
200
0
0
nmoles nitrite reduced.
h-1. mg protein-1
1
c
800
700
600
500
400
300
200
100
0
0
Glutamine Concentration (mM)
Fig. 6. The effect of varied glutamine concentrations on (a) glutamate
formation, (b) 1-14CO2 release, and (c) nitrite reduction by root plastids.
Plastids were supplied with 1 mM NO
2 , 5 mM 2-oxoglutarate, and
either 0.5 mM (filled diamonds) or 5 mM (filled squares) [1-14C]Glc6P. Each value represents the mean 6SEM from at least three
independent experiments.
GOGAT, implying that the OPPP is not producing
sufficient reductant to support the optimal activities of
these two enzymes simultaneously. One way in which the
supply of reductant may be restricted is by the activity of
enzymes of the OPPP. Previously the activity of 6PGDH
in pea root plastids has been shown to increase significantly when roots are incubated in nitrate prior to
harvesting (Emes and Fowler, 1983). Glucose-6-phosphate dehydrogenase (G6PDH) is also part constitutive
and part induced and it seems that the activity of G6PDH
is closely linked to the activities of the inducible nitrogen
assimilatory enzymes (Emes and Fowler, 1979; Redinbaugh and Campbell, 1998). G6PDH activity in plastids
isolated from pea roots grown in the absence of nitrate
was 2556 nmol mg1 protein h1. This increased with the
nitrate concentration supplied to intact roots 24 h prior to
harvesting, reaching a maximum activity of 8150 nmol
In roots, nitrate assimilation is dependent upon a supply of
reductant initially generated by oxidative metabolism, a
significant portion of which is generated by the OPPP. Nitrate
is reduced to nitrite in the cytosol. The uptake of nitrite into
the plastids and its subsequent reduction by nitrite reductase
and glutamate synthase are potentially important control
points. The reduction of nitrite and synthesis of glutamate
within the plastid are dependent upon both the uptake of
Glc6P and its subsequent oxidation. As such, either of these
processes could limit the capacity for nitrogen assimilation.
In this paper the relative importance of Glc6P transport and
the OPPP in controlling nitrogen assimilatory enzymes
dependent on oxidative metabolism in root plastids has been
examined.
Pea root plastids are able to transport glucose-6phosphate in counter exchange with Pi, DHAP, or 3PGA
(Borchert et al., 1989, 1993). The Vmax value for Glc6P
uptake, using the release of 32P-labelled Pi to measure
back exchange, was measured at 0.51 lmol mg1 protein
h1. Transport of Glc6P across the plastid membrane
could limit flux of hexose phosphate through the oxidative
pentose phosphate pathway. DIDS was found to be
a potent inhibitor of Glc6P-dependent nitrite reduction
(Fig. 1). The nature of the covalent modification means it
was not possible to measure the direct effects of DIDS on
Glc6P uptake, therefore this experiment was repeated
using known physiological competitive inhibitors of
Glc6P uptake, Pi, PGA or DHAP (Fig. 2). It was found
that even when approximately 50% of Glc6P uptake was
inhibited by Pi, PGA, or DHAP, sufficient Glc6P was imported to support nitrite reduction (Fig. 3). Although these
experiments are in vitro studies, it seems likely that
control over Glc6P-dependent nitrite reduction is not
through the transport of Glc6P. Since the GPT catalyses
the counter exchange of Glc6P and DHAP, it is perhaps
not surprising that the GPT exerts such little control over
Glc6P-dependent nitrite reduction. A high control coefficient for Glc6P transport would mean that the process
of nitrite reduction within the organelle could otherwise
be severely limited by the presence of Pi, triosephosphate,
and PGA in the cytosol.
The uptake of inorganic nitrite into the plastids is
another possible point of regulation of Glc6P-dependent
nitrite reduction. Nitrite moves from the cytosol to the
plastid where it is reduced by NiR to ammonium. Since
nitrite is toxic, the intracellular concentration is kept at
a low level in normal circumstances through a combination
1116 Bowsher et al.
of NR expression, NR catalytic activity, and NR protein
degradation (Kaiser and Huber, 2001). When the nitrite
reducing capacity is low, protein phosphorylation inactivates NR (Kaiser and Huber, 2001). Nitrite may be
transported freely in its protonated form as nitrous acid or
as an ion. In plants the molecular mechanism of nitrite
uptake into plastids is unclear and there is conflicting data
regarding the presence of a specific nitrite transporter
(Brunswick and Cresswell, 1988; Shingles et al., 1996). In
Chlamydomonas reinhardtii, the unicellular eukaryotic
and photosynthetic green alga, plastidic nitrite transporters
allowing the entry of nitrite into the chloroplasts have
been identified (Mariscal et al., 2004; Rexach et al.,
2000). To date, no potential nitrite transporters have been
identified in higher plants. In the experiments reported
here the availability of 13N-labelled nitrite has meant that
it has been possible to perform plastid labelling studies
and determine uptake of nitrite into plastids over a range
of nitrite concentrations. It is clear from Fig. 4 that nitrite
enters the root plastids by diffusion and as such is unlikely
to regulate Glc6P-dependent nitrite reduction. Indeed, its
transport across the plastid envelope increases if nitrite is
being reduced by NiR (data not shown).
It has previously been shown that within pea root
plastids, the oxidation of Glc6P by the OPPP is capable of
supporting the activity of NiR (Bowsher et al., 1989) and
GOGAT (Bowsher et al., 1992). Furthermore, from the
studies reported here, under substrate saturating conditions, NiR and GOGAT compete for reductant-generated
by the OPPP (Fig. 5). The restriction on Glc6P-dependent
nitrogen metabolism appears to be due to the rate of
supply of reductant as opposed to the ability of enzymes
of nitrogen assimilation to utilize it. The reductant supply
may be restricted in a number of possible ways. Of all the
OPPP enzymes, it is G6PDH which is usually found to
have the lowest maximal activity (Emes and Fowler,
1983). Depending on status of nitrate induction, pea root
G6PDH activity was 2556–8150 nmol mg1 protein h1.
To support the maximum rate of nitrite reduction of 1000
nmol mg1 protein h1 (Fig. 5b), 1500 nmol mg1
protein h1 Glc6P would need to be oxidized. To support
the maximum rate of glutamate formation of 1500 nmol
mg1 protein h1 (Fig. 5c), 375 nmol mg1 protein h1 of
Glc6P would need to be oxidized. The total amount of
Glc6P which would need to be oxidized to support both
nitrite reduction and glutamate synthesis under saturating
conditions is thus 1875 nmol mg1 protein h1, indicating
that the capacity of G6PDH is more than sufficient to
meet this demand and nearly 6-fold in excess when fully
induced. G6PDH has been reported to be strongly
inhibited by a high NADPH/NADP ratio (Wright et al.,
1997). Studies on isolated barley root plastids suggest that
it is the P2 isoform of G6PDH which is present in root
plastids (Esposito et al., 2001a). This isoform appears
able to continue to support reactions under NADPH/
NADP ratios that would strongly inhibit the chloroplastic
and cytosolic isoforms of the enzyme (Wright et al., 1997;
Esposito et al., 2001a) indicating that the ratio of reduced
to oxidized pyridine nucleotide is also unlikely to be
a limitation.
The oxidation of 1 mol of Glc6P via G6PDH and
6PGDH generates four electrons (2 NAPDH). To achieve
electron transfer efficiently from the OPPP to the enzymes
of nitrite assimilation under saturating conditions, 1870
nmol mg1 protein h1 would require FNR to operate at
a rate of 7632 nmol mg1 protein h1. Previously, it has
been reported that an FNR activity of 9000–18 000 nmol
mg1 protein h1 was in root plastids isolated from plants
induced with 10 mM nitrate (Bowsher et al., 1993). This
rate was determined using a non-physiological cytochrome c-based assay and represents a one electron
transfer. Although the maximum rate of transfer of
NADPH to ferredoxin is not known, if it is assumed to be
equivalent to the rate of cytochrome c reduction, FNR
may not limit flux through the OPPP. However, since this
is only slightly in excess of the rate required to support
nitrite reduction and glutamate synthesis, it is an area
worthy of further investigation. An alternative point of
control may be competition between NiR and GOGAT for
reduced ferredoxin. Yonekura-Sakibara et al. (2000)
found that maize roots contained 0.41 lg (approximately
410 nmol) mg1 protein ferredoxin, and 1.08 lg (approximately 180 nmol) mg1 protein sulphite reductase
(another ferredoxin-dependent bioassimilatory enzyme).
Such equivalent concentrations of the electron donor and
only one of its dependent enzymes raises the possibility
that access to unbound, reduced ferredoxin may limit the
activity of several enzymes of reductive assimilation,
including NiR and GOGAT.
The results described here suggest that the restriction on
Glc6P-dependent nitrogen metabolism in root plastids is
due to the rate of supply of reductant as opposed to the
ability of the enzymes of nitrogen assimilation to accept
reductant. Competition for electrons between nitrite reductase and glutamate synthase imply that the OPPP is not
capable of producing sufficient reductant to support the
optimal activities of these two enzymes simultaneously.
Although NiR and GOGAT represent different sink
strengths needing six electrons and two electrons per
reaction, respectively, they inhibit each other approximately in proportion and the percentage reduction in
activity during competition for reductant is approximately
the same for each enzyme (Fig. 7). For example, at a
concentration of 1 mM Glc6P, nitrite reduction is decreased by approximately 20% in the presence of
saturating glutamine and 2-oxoglutarate. Likewise, at the
same Glc6P concentration, GOGAT activity is decreased
by 20% in the presence of saturating nitrite. This suggests
that the oxidation of Glc6P through the OPPP may place
a significant constraint on the reactions of nitrogen
100
100
80
80
60
60
40
40
20
20
0
0
2
4
6
8
% difference in nitrite
reduced
% difference in glutamate
formation
Glc6P uptake and oxidation in pea root plastids 1117
10
Glc6P Concentration (mM)
Fig. 7. Comparison of the percentage decrease in glutamate synthesized
(filled diamonds) and nitrite reduced (filled squares) in pea root plastids
when there is competition between NiR and GOGAT for reductant
produced by the OPPP. Glc6P-dependent nitrite reduction (100%¼1000
nmol mg1 protein h1) and Glc6P dependent-glu formation
(100%¼1481 nmol mg1protein h1) were measured in pea root
plastids incubated for 60 min with different concentrations of Glc6P
and 1 mM NO
2 , 5 mM Gln+5 mM 2-oxoglutarate.
assimilation in plastids. In addition, there may be
a mechanism for partitioning of electrons between the
two enzymes so that they remain in balance. Such
partitioning may be essential to the functioning of
nitrogen assimilation in vivo. An additional consideration
must be given to species where the root is the principal
site of nitrate reduction. In Lotus japonicus, for example,
as with many other temperate legumes, there must be
a heavy demand for electrons from carbohydrate oxidation
which extends throughout the diurnal cycle (Prosser et al.,
2006).
Nitrate assimilation is a highly regulated and integrated
metabolic process (Stitt, 1999). It is dependent on a range
of signals including the amount and type of nitrogen
source, light and carbon availability. The complex nature
of this regulation reflects the multiple levels of control.
Non-photosynthetic plastids are involved in a range of
other metabolic processes including fatty acid synthesis. If
other pathways are superimposed onto the reductant
demands of nitrogen assimilation, carbohydrate oxidation
within plastids is likely to become even more limiting for
any given activity and thus play an important role in
regulating assimilatory and biosynthetic pathways.
Acknowledgements
The authors are grateful to the BBSRC, MWO, and Royal Society
for financial support. They also gratefully acknowledge the PET
Center, Groningen, for providing 13N.
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