Planta (1995)196:58-63
P l m a t ~
9 Springer-Verlag 1995
Phosphoenolpyruvate carboxykinase from higher plants:
Purification from cucumber and evidence of rapid
proteolytic cleavage in extracts from a range of plant tissues
Robert P. Walker, Stephen J. Trevanion, Richard C. Leegood
Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield,Sheffield,$10 2UQ, UK
Received: 10 August 1994 / Accepted: 7 September 1994
Abstract. Phosphoenolpyruvate carboxykinase (PEPCK)
was purified 600-fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.) and a polyclonal
antiserum raised. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the purified
preparation contained a single polypeptide of 62 kDa,
consistent with previous studies of this enzyme in C4
grasses. Immunoblots of crude extracts showed that a
form of P E P C K of approximately this molecular mass
predominated in cucumber cotyledons and in a range of
plant tissues (cotyledons of fat-storing seedlings, leaves of
C4 and Crassulacean acid metabolism plants). However,
when these tissues were extracted in the presence of SDS
and the extracts analysed by immunoblotting, a larger
polypeptide of 68-77 kDa was detected. Thus the enzyme
generally measured in crude extracts is a smaller form
which arises by rapid proteolysis. This phenomenon
means that the native form of PEPCK has never been
purified from plants nor its properties determined.
Key words: C4 plant - Crassulacean acid metabolism Cucumis - Phosphoenolpyruvate carboxykinase - (Purification) - Proteolytic degradation
Introduction
In plants, phosphoenolpyruvate carboxykinase (PEPCK,
EC 4.1.1.49) catalyses a key step in the conversion of fats
to sugars during the germination of fat-storing seeds, in
which its activity closely parallels the gluconeogenic flux
(Leegood and ap Rees 1978). It also plays an important
role in photosynthetic carbon assimilation in some
Abbreviations: CAM = Crassulacean acid metabolism; DTT =
dithiothreitol; PEG = polyethyleneglycol; PEP = phosphoenolpyruvate, PEPCK = phosphoenolpyruvate carboxykinase;
Rubisco = ribulose-l,5-bisphosphate carboxylase-oxygenase
Correspondence to: R.C. Leegood; FAX: 44 (1 t4) 2760159; Tel.: 44
(114) 2824780; E-mail: r.leegood@sheffield.ac.uk
plants, including partial decarboxylation of C4 acids in
one group of C4 plants and in Crassulacean acid
metabolism (CAM) plants such as the bromeliads (Leegood and Osmond 1990; Carnal et al. 1993), as well as a
role in the CO2-concentrating mechanism of certain algae
(Holdsworth and Bruck 1977; Weidner and Ktippers
1982; Reiskind et al. 1988; Reiskind and Bowes 1991). In
addition, it plays a role in the ripening of grapes (Ruffner
and Kliewer 1975).
Phosphoenolpyruvate carboxykinase also occurs
widely in animals, fungi and bacteria. There are, however,
two types of the enzyme. One type, from mammals, birds
and insects, utilises either guanine or inosine nucleotides
as a cofactor and has a molecular mass of about 70 kDa.
The sequences have a high degree of homology (Reymond et al. 1992). The other type of PEPCK, from higher
plants, bacteria, yeast and trypanosomes, preferentially
utilises adenine nucleotides. In higher plants, the enzyme
has been purified to homogeneity from the leaves of three
C4 grasses (Burnell 1986) and partially purified from
cotyledons of marrow (Cucurbita pepo) (Leegood and ap
Rees 1978) and from the leaves of the CAM plant,
pineapple (Daley et al. 1977). The study by Burnell (1986)
showed the enzyme to be a hexameric protein, each subunit with a molecular mass of 64 kDa, which is lower
than that of the animal enzyme but is similar to, or slightly larger than, PEPCK from yeast (64 kDa; Tortora et al.
1985), Escherichia coli (50-55 kDa; Goldie and Sanwal
1980), Rhizobium (58 kDa; Ostergts et al. 1991) and Trypanosoma brucei (60 kDa; Kueng et al. 1989; Parsons and
Smith 1989). Recently, a clone for PEPCK has been isolated from a eDNA library from senescing cucumber
cotyledons. The gene codes for a protein which is between 43 and 57% identical to the bacterial (E. coli and
Rhizobium), yeast and trypanosomal PEPCKs at the
amino-acid level, including a conserved ATP-binding domain. There are no sequence homologies with PEPCK
from animals. However, the molecular mass of cucumber
PEPCK predicted from the eDNA sequence is about
74 kDa, and a polyclonaI antiserum raised to the protein
produced when a portion of the cDNA was over-ex-
R.P. Walker et al. : Phosphoenolpyruvate carboxykinase from higher plants
p r e s s e d in E. coli r e c o g n i s e d a 7 4 - k D a p r o t e i n in c u c u m b e r e x t r a c t s ( K i m a n d S m i t h 1994).
T h e a i m o f this w o r k w a s t o p u r i f y P E P C K f r o m cuc u m b e r c o t y l e d o n s to h o m o g e n e i t y a n d to raise a p o l y c l o n a l a n t i s e r u m . T h e results s h o w t h a t P E P C K f r o m a
r a n g e o f tissues o f h i g h e r p l a n t s is i n d e e d a l a r g e r p r o t e i n
t h a n e a r l i e r w o r k s u g g e s t e d a n d t h a t the e n z y m e g e n e r a l ly m e a s u r e d in c r u d e e x t r a c t s is a p r o t e o l y t i c a l l y d e g r a d e d f o r m o f t h e e n z y m e w h i c h exists in v i v o .
59
pended in a minimum volume of 2 0 m M 2[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-l,3-propanediol (Bistris) propane
(pH 6.5), 5 mM DTT, 20% (v/v) glycerol (buffer C).
Mono Q chromatography. A 1-ml sample was applied to a Mono Q
HR5/5 (Pharmacia, Milton Keynes, UK) fast protein liquid chromatography (FPLC) anion-exchange column at a flow rate of
0.25 ml 9min -1. The column was washed with 10 column volumes
of buffer C at a flow rate of 1 ml. m i n - 1. Bound protein was eluted
by applying a 0-250 mM linear gradient of NaC1.
Protein determination. Protein was determined using a modified
Materials and methods
Plant material. Seeds of cucumber (Cucumis sativus L. cv. Masterpiece; W. McNair, Portobello, Edinburgh) were imbibed in distilled
water for 4 h at 25 ~ C and sown in moist vermiculite. The seedlings
were either grown in the light (350 lamol quanta, m - 2 . s -1) or in
the dark at a constant temperature of 25 ~ C. For enzyme purification, seeds were germinated and grown at 25 ~ C in the dark for 5 d.
Seeds of oil seed rape (Brassica napus L. biennus), peanut (Arachis
hypogaea L.), marrow (Cucurbita pepo L. cv. Green Bush), sunflower
(Helianthus annuus L.) and nasturtium (Tropaeolum majus L.) were
grown in soil for 5 d at 25 ~ C in darkness. Urochloa panicoides and
Nidulariumfulgens were grown in a greenhouse under ambient light.
Tissues were either used fresh or after freezing in liquid N2 and
storage at - 8 0 ~ C.
Extraction and assay of PEPCK. Plant material was homogenised
in a mortar with 10 volumes of ice-cold extraction buffer A
[200 m M N,N-bis(2-hydroxyethyl)glycine (Bicine) K O H (pH 9.0),
20 mM MgC12, 5 m M dithiothreitol (DTT), then clarified by centrifugation at 12 000"g for 5 min. The carboxylation reaction of
PEPCK was assayed according to Cooper et al. (1968) at 25 ~ C.
The assay contained, in 1 ml: 80 mM 2-(N-morpholino) ethanesulfonic acid (Mes)-NaOH (pH 6.7), 0.35 mM N A D H , 5 mM DTT,
2 mM MnC12, 2 mM phosphoenolpyruvate (PEP), 2 mM ADP,
50 mM KHCO3, 6 U malate dehydrogenase. This assay was used in
crude extracts and throughout the purification. The decarboxylation assay was measured in a stopped reaction. The assay contained, in 800 lal: 40 mM Hepes-KOH (pH 8.0), 0.25 m M ATP,
0.5 m M MnC12, 0.1 mM oxaloacetate. After 10 min at 25 ~ C reactions were stopped by heating to 100~ for 2 min. The PEP was
determined by adding an aliquot of the reaction mixture to 30 mM
K2HPO4/20 m M NaH2PO 4 (pH 7.0), 10 mM MgC12, 1 mM ADP,
1.5 mM NADH, 6 units lactate dehydrogenase and initiating the
reaction with 0.5 units pyruvate kinase.
version of the procedure described by Lowry et al. (1951), adapted
for a micro-titre plate (Fryer et al. 1986). Proteins were precipitated
by TCA, resuspended in 2% (w/v) Na2CO 3, 0.1 M NaOH, 1% (w/v)
SDS and incubated at 37 ~ C for 1 h. After dissolution of the pellets,
aliquots were assayed for protein. Bovine serum albumin was used
as a standard.
Analysis by SDS-PAGE. Protein samples were added to solubilisation buffer (10% glycerol, 5% SDS, 5% 2-mercaptoethanol, 0.002%
bromophenol blue, 62.5 m M Tris-HCl, pH 6.8), placed at 100 ~ C for
3 min and centrifuged at 13 000"g for 3 min. For SDS-PAGE
(Laemmli 1970), a 4.7% T/2.7% C stacking gel and a 10.5% T/2.7%
C resolving gel were used. After electrophoresis, polypeptides were
fixed in gels by immersion in 50% (v/v) methanol and 12% (v/v)
acetic acid. Polypeptides were visualised by colloidal Coomassie
blue G-250 (Sigma, Poole, Dorset, UK) and then counterstained
with silver (Rabilloud et al. 1988).
Preparation of an antiserum to PEPCK. A polyclonal antiserum was
generated in a New Zealand rabbit. 250 lal of the purified preparation, containing 200 ~tg of protein, was mixed with an equal volume
of Freund's complete adjuvant and injected subcutaneously at four
sites on the rabbit. Booster injections, each containing 200 tag
protein, were done four and six weeks later using Freund's incomplete adjuvant. Two weeks after the final boost, the rabbit was bled.
The blood was allowed to clot and the serum obtained was stored
at - 2 0 ~ C. An antiserum to rape ribulose-l,5-bisphosphate carboxylase-oxygenase (Rubisco) was generated by the same procedure.
Immunoblotting. Transfer of polypeptides from an SDS-PAGE gel
to a nitrocellulose membrane was done in a Pharmacia Multiphor
apparatus. Immunoreactive polypeptides were visualized using a
peroxidase-conjugated second antibody.
Purification of PEPCK. All procedures except Mono-Q chromatog-
Results and discussion
raphy were carried out at 0 ~ ~ C. Cotyledons were homogenised in
a mortar containing 2 volumes of extraction buffer B [50 mM 1,4piperazinediethanesulfonic acid (Pipes)-NaOH (pH 6.7), 5 mM EDTA, 5 mM DTT, 20 mM MnC12, 10 mM MgC12, 400 mM sucrose].
Purification o f P E P C K . W h e n P E P C K h a d b e e n p u r i f i e d
Ammonium sulfate fractionation. The homogenate was clarified by
successive centrifugations at 2500 9g for 10 min and 25 000 9g for
20 min. Protein in the supernatant that precipitated between 40 and
50% saturation with ( N H 4 ) 2 S O 4 w a s collected by centrifugation at
10 000 - g for 15 min. The pellet was resuspended in a minimal volume of 50 mM Pipes-NaOH (pH 6.7), 5 m M DTT and 10 mM MgC12.
Acid precipitation. Four volumes of 300 mM Na-acetate (pH 4.2)
containing 5 mM DTT were added to the preparation, mixed and
left for 30 min. Insoluble material was then removed by centrifugation at 10 000 9 g for 15 min. Polyethylene glycol 6000 (PEG; 40%)
in 50 mM Pipes-NaOH (pH 6.7), 5 mM DTT and 10 mM MgC12
was added to the supernatant to a final concentration of 15% (w/v)
PEG. After incubation for 30 min, precipitated protein was collected by centrifugation at 10 000 - g for 15 min. The pellet was resus-
6 0 0 - f o l d t h e p r e p a r a t i o n p o s s e s s e d a s i m i l a r specific act i v i t y ( T a b l e 1) to t h a t o f t h e p u r e e n z y m e i s o l a t e d f r o m
t h e l e a v e s o f C4 g r a s s e s ( B u r n e l l 1986). A f t e r S D S - P A G E ,
t h e p r e p a r a t i o n c o n t a i n e d a single p o l y p e p t i d e w h i c h
w a s o f a s i m i l a r size (62 k D a ) t o t h a t o f P E P C K f r o m C4
g r a s s e s ( B u r n e l l 1986). A m i n o - a c i d s e q u e n c i n g o f a p e p tide produced by cyanogen bromide cleavage of the purified p r o t e i n s h o w e d a s e q u e n c e o f 12 a m i n o a c i d s i d e n t i cal t o t h a t d e d u c e d f r o m t h e c D N A for P E P C K ( K i m
a n d S m i t h 1994), c o n f i r m i n g t h a t this p r o t e i n w a s
PEPCK. Gel-filtration chromatography of the purified
p r e p a r a t i o n s h o w e d it to be a t e t r a m e r ( a p p r o x . 270 k D a )
s u g g e s t i n g t h a t , in c o m m o n w i t h P E P C K f r o m C 4 g r a s s e s
( h e x a m e r ; B u r n e l l 1986), y e a s t ( t e t r a m e r ; T o r t o r a et al.
1985) a n d Trypanosoma cruzi ( d i m e r ; U r b i n a 1987), t h e
cucumber enzyme was a multimer. Whether or not these
60
R.P. Walker et al.: Phosphoenolpyruvate carboxykinase from higher plants
Table 1 Purification of the 62-kDa form of PEP CK from 160 g of cucumber cotyledons. The enzyme was extracted and purified as described
in Materials and methods
Stage
Total activity
(lamol ' min- 1)
Protein
(mg)
Specific activity
Purification
(U. rag- 1 protein) ( - fold)
Yield
(%)
Crude extract
Soluble fraction
40-50% (NH4)2SO 4
Acid precipitation
PEG precipitation
Mono-Q chromatography
1270
1186
724
720
356
235
15180
8433
749
128
19
4.9
0.08
0.14
0.97
5.6
18.7
48,0
100
93
57
57
28
19
74 kDa --)
62 kDa ---)
Fig. 1. Western blot showing varying stability of PEPCK in extracts
of cucumber cotyledons. Cotyledons were homogenised in extraction buffer B at pH 6.7 (see Materials and methods) alone or in the
presence of 1% SDS and left at
25~ C for 30 rain. The integrity of
PEPCK was assessed by immunoblot analysis of polypeptides
separated by SDS-PAGE, using
an antiserum specific for PEPCK
differences in aggregation state are a consequence of the
degradation of the enzyme (see below) is difficult to assess
at this stage.
The activity of P E P C K was stable for several months
when the purified preparation was stored at - 20 ~ C. The
presence of D T T was essential at all stages of the purification to prevent loss of activity (see also Ray and Black
1976; Burnell 1986). Kinetic studies using purified enzyme showed that it possessed similar kinetic properties
to those of P E P C K s isolated from other plant tissues
(Daley et al. 1977; Leegood and ap Rees 1978; Burnell
1986). Adenine nucleotides were preferred for both carboxylation and decarboxylation reactions. The Michaelis
constants, determined from Eadie-Hofstee plots, for the
carboxylation reaction were: P E P 0.34 mM, A D P 27 laM
and CO 2 16 m M ; and for the decarboxylation reaction
were: oxaloacetate 17 laM and A T P 14 gM.
Forms o f P E P C K in vivo. A polyclonal antiserum was
raised against the purified P E P C K and was used to probe
Western blots of S D S - P A G E gels. These blots showed
that the antiserum recognised specifically the P E P C K
polypeptide at all stages of the purification. However,
further investigation revealed two forms of PEPCK. One,
with a molecular mass of a b o u t 74 k D a was observed
when cotyledons were extracted in buffer at p H 6.7 in the
1
1.8
12
70
234
600
Fig. 2. Western blot showing varying stability of PEPCK in extracts
of cucumber cotyledons homogenised in extraction buffer A (see
Materials and methods) at either pH 7.0 or pH 9.0. After homogenisation, extracts were left at 25~ C for up to 90 min. The integrity of
PEPCK was then assessed by immunoblot analysis of polypeptides
separated by SDS-PAGE
presence of SDS. The other, 62-kDa, form was observed
when extracts were prepared in buffer at pH 6.7 and left
for short periods before denaturation (Fig. 1). These results imply that a protease rapidly cleaves the 74-kDa
form of P E P C K to generate the 62-kDa form. A wide
range of protease inhibitors, including leupeptin, pepstatin, antipain, chymostatin, 4-amidinophenylmethanesulfonyl fluoride (APMSF), bestatin, aprotinin and E64, had
no effect on cleavage of P E P C K in crude extracts at p H
6.7. We then investigated whether cleavage of the 74-kDa
form could be prevented under any conditions. Extraction at a more-alkaline p H (pH 9.0) was shown to result
in enhanced stability of the 74-kDa form (Fig. 2), for up
to 90 min, suggesting that the protease is inactive at this
high p H value.
The rapid proteolytic cleavage of P E P C K in extracts
of cucumber cotyledons raises the question of the extent
to which this is a general phenomenon. As pointed out in
the Introduction, P E P C K from other plants, such as C4
grasses, has been shown to have a molecular mass of
around 62 kDa. We investigated whether the rapid proteolytic cleavage observed in extracts of cucumber cotyledons was also a feature of other plant tissues. First, we
studied a range of tissues close to the peak of gluconeogenesis. This antiserum to cucumber P E P C K possessed a
similar affinity for P E P C K from a range of plant tissues.
Oil seed rape, peanut and m a r r o w (Fig. 3) and sunflower
and nasturtium (data not shown) all showed similar features to cucumber, with a rapid proteolytic cleavage from
R.P. Walker et al.: Phosphoenolpyruvate carboxykinase from higher plants
61
Fig. 3. Western blot showing varying stability of PEPCK in extracts
of cotyledons of a range of fat-storing seedlings. Cotyledons were
homogenised in either extraction buffer A (see Materials and methods) at pH 9.0 containing 1% SDS and boiled immediately or in
extraction buffer A at pH 7.0 and incubated at 25~ C for 30 rain
Urochloa panicoides
Nidularium fulgens
Fig. 5. Western blot showing stability of the stromal fructose-l,6bisphosphatase and Rubisco in extracts of cucumber cotyledons
homogenised in extraction buffer A at pH 7.0. After homogenisation, extracts were left at 25~ for up to 90min. Changes in
polypeptides were monitored by SDS-PAGE and immunoblotting
using antisera specific either for fructose-l,6-bisphosphatase or Rubisco
i8 kDa-->
~2 kDa-->
fulgens prepared at pH 7.0 also showed degradation of a
9
+ SDS
7
-SDS
9
7
+ SDS -SDS
extraction conditions
Fig. 4. Western blot showing varying stability of PEPCK in homogenates of leaves of the C4 grass, Uroehloa panicoides and of the
CAM bromeliad, Nidulariumfulgens. Leaves were homogenised in
either extraction buffer A (see Materials and methods) at pH 9.0
containing 1% SDS and boiled immediately or in extraction buffer
A at pH 7.0 and incubated at 25~ C for 30 min. The integrity of
PEPCK was then assessed by immunoblot analysis of polypeptides
separated by SDS-PAGE
a 74-kDa form to a 62-kDa form at lower p H values (pH
7.0) in the absence of SDS. Second, we examined extracts
made from the leaves of the P E P C K - t y p e C4 grass,
Urochloa panicoides, and the C A M bromeliad, Nidularium fulgens (Fig. 4). Extracts of U. panicoides showed
rapid degradation of P E P C K from a 68-kDa form to a
62-kDa form at neutral pH, which was prevented by extraction at pH 9.0 in the presence of SDS. Extracts of N.
77-kDa form, but in this case there were a number of
minor bands generated around 60 kDa, rather than than
a clearly defined major band at 62 k D a as found in the
other tissues. Examination of previous purification protocols shows that several resulted in multiple forms eluting from ion-exchange columns (e.g. Daley et al. 1977;
Leegood and ap Rees 1978).
Western blots also showed that P E P C K in extracts of
cucumber roots was rapidly degraded (data not shown),
although the a m o u n t and activity of P E P C K in roots
was, on a fresh-weight basis, but a small fraction (less
than 2%) of the activity in cotyledons.
We also investigated whether any other enzymes
might be subject to rapid proteolysis in extracts of cucumber cotyledons. Neither Rubisco nor the stromal
fructose-l,6-bisphosphatase was affected by incubation
at pH 7.0 in extracts of greened cucumber cotyledons
(Fig. 5). The total polypeptide pattern as seen on SDSP A G E gels was also not visibly affected by incubation of
the extracts for prolonged periods at p H 7.0 (data not
shown). These limited data suggest that the proteolytic
activity leading to the cleavage of P E P C K was not entirely promiscuous (compare Usuda and Shimogawara
1994).
62
R.P. Walker et al.: Phosphoenolpyruvate carboxykinase from higher plants
Fig. 6. Changes in PEPCK protein, as revealed by SDS-PAGE and immunoblotring, and PEPCK activity in cucumber
cotyledons following imbibition by the
seed and subsequent growth in darkness
at 25~ C. Protein was extracted in extraction buffer A (see Materials and methods)
at pH 9.0 containing 1% SDS and boiled
immediately. Activity was measured as
described in Materials and methods after
extraction at pH 9.0
The fact that this cleavage of P E P C K occurs in a wide
range of tissues, both gluconeogenic and non-gluconeogenic, suggests that it is unlikely to serve the same physiological function. We checked whether the 62-kDa form
of P E P C K was present in cucumber cotyledons at any
stage of development. Figure 6 shows Western blots from
gels of extracts prepared from the onset of germination
through the peak of gluconegenic activity. At no stage
was there any evidence for the presence of the 62-kDa
form of the enzyme in either dark-grown seedlings
(Fig. 6) or light-grown seedlings (data not shown). From
these data we conclude that the 62-kDa form of P E P C K
does not exist in vivo and is entirely artefactual.
There have been m a n y measurements of P E P C K activity reported for a range of tissues. The observation that
the enzyme is rapidly cleaved then raises the question of
whether cleavage leads to any loss of activity of PEPCK.
To test this we t o o k cotyledons and extracted them at
both p H 7.0 and p H 9.0 (as in Fig. 2). Both were then
assayed spectrophotometrically at p H 6.7. The assay under these conditions was linear immediately after the addition of the extract and the activities were identical for
both extracts. These data suggest that cleavage leads to
no loss of activity. Figure 6 shows activities of P E P C K
measured after extraction at p H 9.0 and assay at p H 7.0.
Both the pattern of activity during germination and the
absolute activities strongly resemble those already determined in m a r r o w cotyledons (Leegood and ap Rees 1978)
and they also resemble the pattern of changes observed
for other gluconeogenic enzymes in cucumber (Trelease
et al. 1971; Becker et al. 1978). Nevertheless, this phen o m e n o n of rapid proteolysis means first, that P E P C K
has never been purified from plant tissues in its native
form. Second, it follows that the properties of P E P C K
have never been adequately determined in crude extracts
of plant tissues, in spite of the fact that the cotyledons of
fatty seedlings have long been recognised as a particularly amenable tissue for the assay of enzymes. The work
demonstrates the importance of using antibodies to
check for such degradation in crude extracts of plant
tissues. It should be noted that there are now a number of
examples in the literature where rapid proteolytic cleavage has been observed in crude extracts, e.g. with Rubisco
(Usuda and Shimogawara 1994); ADPglucose pyrophosphorylase (Plaxton and Preiss 1987; Kleczkowski et al.
1993), N A D P - m a l a t e dehydrogenase (Kampfenkel 1992)
and PEP carboxylase (Baur et al. 1992; Usuda and Shim o g a w a r a 1994), often without affecting Vm,x.
The observation that the 74-kDa form of P E P C K
could be stabilised at high p H values has now enabled us
to develop a method for the purification of the 74-kDa
form of P E P C K and to investigate its regulation.
We are grateful to Dr. Steve Smith (University of Edinburgh, UK)
for helpful discussions, Dr. Alf Keys (Institute of Arable Crops
Research, Rothamsted, UK) for the gift of pure Rubisco and Dr.
Tristan Dyer (John Innes Centre for Plant Science Research, Norwich, UK) for the antiserum to fructose-l,6-bisphosphatase. This
research was supported by the joint Agricultural and Food Research Council / Science and Engineering Research Council Programme on the Biochemistry of Metabolic Regulation in Plants
(PG50/590).
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