From the
Metabolic pathways of the intermediate metabolism of maize root
tips were identified and quantified after labeling to isotopic and
metabolic steady state using glucose labeled on carbon-1, -2, or -6
with
Non-photosynthetic cells of higher plants depend on the supply
of a carbon substrate, usually sucrose provided by leaves or storage
organs, for their metabolism. Sucrose is converted to sugar phosphates,
which can be used for the biosynthesis of structural or storage
carbohydrates, or degraded by respiration to produce ATP and
intermediates for biosynthesis
(1) . In recent years, specific
aspects of the metabolism of non-green cells have been described
semi-quantitatively by label distribution studies using classical
techniques of radiolabeling or NMR methods for metabolite analysis. In
most plant tissues studied, cycling between hexose phosphates and
sucrose was indicated by the labeling of both glucose and fructose, and
of the hexosyl moieties of sucrose, from labeled glucose or fructose
(2, 3). The occurrence of a futile cycle between hexose phosphates and
triose phosphates was indicated by the randomization of label between
C-1 and C-6 observed in sucrose, starch, and
hexoses
(4, 5, 6) , and an activity of the
pentose phosphate pathway was deduced from the different randomization
from C-1 to C-6 than from C-6 to C-1
(4, 5, 7) .
In addition, the limited randomization observed in starch indicated
that, in non-green tissues, hexose phosphates rather than triose
phosphates enter the plastid for starch
synthesis
(4, 5, 6) . Fluxes were calculated from
specific yields or from randomization
(5, 7) using
simple models of glycolysis and the pentose phosphate pathway, which
assumed complete recycling of fructose phosphate and a unidirectional
transaldolase reaction. However, recycling may be partial, and the
transaldolase reaction is reversible
(8, 9) . These
simple models may thus underestimate the flux into the pentose
phosphate pathway and overestimate the conversion of triose phosphate
to hexose phosphate. Fluxes of carbon input into the tricarboxylic acid
cycle, including the partition of the glycolytic flux at the
phosphoenolpyruvate (PEP)
Root tips of maize (Zea mays L.) are
commonly used as a model for studies of energy metabolism in
non-photosynthetic tissues. Their high metabolic activity makes them
sensitive to limitations of oxygen or carbohydrate supply, which may
determine the survival or death of the tissues. In an early work, based
on the comparison of
The aim of the present work was to identify and quantify
the major metabolic fluxes of carbon metabolism of the maize root tip
to gain a better understanding of its response to stress. We labeled to
isotopic steady state with [
The specific radioactivity of C-1 of glucose (either free glucose or
glucose from starch hydrolysis) was determined after decarboxylation.
The reaction was performed, according to Ref. 31, in a Warburg vial
containing 2 ml of 70 mM sodium phosphate, pH 7.7, 4
mM ATP, 3 mM NADP, 3.8 mM MgCl
The absolute
The labeling of the hexose phosphate C-6,
as shown in sucrose (), may result from the resynthesis of
fructose phosphate from triose phosphates by three ways. (i) The
classical one is the reversal of glycolysis by aldolase and either
fructose-1,6-bisphosphatase or phosphofructophosphotransferase.
Phosphofructophosphotransferase is a particular enzyme present in the
cytosol of higher plant cells, which catalyzes the reversible
phosphorylation of fructose 6-phosphate using pyrophosphate as the
phosphoryl donor
(5) . (ii) The unidirectional transaldolase
reaction of the pentose phosphate pathway incorporates the glucose C-1
present at the C-3 position of triose phosphates into the C-6 position
of fructose phosphate
(8, 9, 13) (preliminary
calculations showed that a reversal of glycolysis sufficient to explain
the high enrichment of hexose phosphate C-6 would produce a C-1
enrichment lower than the experimental one. The incorporation of free
triose phosphate into the fructose phosphate resynthesized through the
unidirectional transaldolase reaction of the pentose phosphate pathway
was also insufficient to account for the enrichment of hexose phosphate
C-6). (iii) The reversibility of the transaldolase reaction
(8) may increase the labeling of hexose phosphate C-6 by
exchange with triose phosphate C-3 without modifying the enrichment of
hexose phosphate C-1. It has usually been ignored in plants because it
does not affect calculations of the pentose phosphate pathway
flux
(9, 13) , but its occurrence was recently suggested
in potato tubers and Vicia faba cotyledons
(6) . All
three pathways were considered for operation in the metabolic network
(Fig. 4).
Given a plastid
location of the pentose phosphate pathway, the cytosolic and plastidial
triose phosphates have to be in rapid exchange to explain the tracer
dilution in alanine and tricarboxylic acid cycle intermediates in
comparison with cytosolic hexose phosphates. Thus, triose phosphates
were assumed to be close to equilibrium, and only one pool was
considered (Fig. 4). This is in agreement with the high activity
of the triose phosphate translocator found in vitro in
non-green pea root plastids
(38) .
The
The enrichment
of glutamate C-4 was identical to that of alanine C-3, thus indicating
the absence of any flux of unlabeled carbon between pyruvate and
glutamate. The
Calculation of glutamate C-4
enrichment from the multiplets of the
The relative flux of sucrose cycling, Suc, was
determined graphically from a plot of Equations 1 and 6 in which the
parameters H1 and H6 were given the two extreme experimental values of
the enrichment of sucrose C-1 (79 and 81%) and sucrose C-6 (13 and
14%), respectively (Fig. 8). The estimated value of Suc was 3.1 ± 0.3 times the flux of glucose entering maize root
tips. The cytosolic fluxes of hexose phosphate resynthesis
(TFc) and of triose phosphate exchange through the cytosolic
transaldolase reaction (Tlc) were determined from Equations 7
and 12, using the Suc values determined above; they were
TFc = 0.38 ± 0.03 and Tlc = 0.25
± 0.1.
The fluxes Pent and Pol correspond to the
synthesis of polysaccharides, which are either incorporated into cell
walls or secreted by the root tip. The sum of the calculated fluxes,
121 nmol hexose
Labeling to isotopic steady state is a classical method for
identifying metabolic pathways and quantifying metabolic
fluxes
(25) . The present work relied mainly on labeling with
[1-
The major
pathways of carbohydrate metabolism in maize root tips were described
quantitatively from the measurement of enrichment of specific carbons
in carbohydrates and in the amino acids glutamate and alanine. No fit
could be obtained with the simplest models, and a relatively
complicated metabolic scheme (Fig. 4) was necessary to account
for the labeling data. The pathways described include sucrose cycling,
different steps of glycolysis, the pentose phosphate pathway, located
in the plastids, and the entries into the tricarboxylic acid cycle. The
validity of the model was confirmed by independent experiments,
including transfer of glucose C-2 to the C-1 position, and measurement
of the increases in insoluble polysaccharides and total proteins, which
were in agreement with the calculated fluxes, thus suggesting that the
model adequately describes the metabolism of the root tip. This may be
surprising in view of the morphologic heterogeneity of the root tip.
Conversely, this may indicate that the metabolic pathways here
described occur in most of the dividing and elongating cells of the
root tip. Differences between cells may occur in pathways not studied
here, or else cells with a significantly different metabolism may be
insufficiently numerous to disturb the results.
It was surprising
that no difference was found in the production of labeled CO
More generally, the
non-triose pathways, which include both the formation of
polysaccharides represented by the sum Pol + Pent (I), and the accumulation of soluble carbohydrates
consumed as much as 74% of the glucose entering the root tips. On the
other hand, the synthesis of starch and lipids represented minor
fluxes. The low contribution of starch to the metabolism of the root
tip is striking compared with most other plant tissues.
The presence
of a pentose phosphate pathway was deduced from the dilution of the
triose phosphate enrichment reflected in alanine and other amino acids,
given that the triose phosphate isomerase was shown to be close to
equilibrium. Its presence was confirmed, and its plastid location was
established, by the transfer of label from C-2 of
[2-
To account for the low enrichment of triose
phosphates and the high enrichment of starch glucosyl units, we had to
consider that only some of the fructose phosphates produced by the
oxidative pentose phosphate pathway were recycled. This flux, A = 0.06 ± 0.02, was 22% of the flux entering the
pentose phosphate pathway instead of 66% in the case of complete
recycling shown in usual models
(5, 7) . This metabolic
feature implies that two distinct fructose phosphate pools are present
inside the plastids (Fig. 4).
There is overwhelming evidence
that in addition to a complete glycolytic pathway in the cytosol,
higher plant cells also contain most, if not all, of the enzymes of the
glycolytic pathway in the plastids. In all the tissues examined so far,
plastids contain the enzymes necessary for the conversion of hexose
phosphates to triose phosphates
(38, 46, 47) .
The reason why only one glycolytic pathway was shown in the metabolic
scheme (Fig. 4) is that it was not possible to decide, from the
experimental data, how much of the total glycolytic flux was located in
the cytosol and how much in the plastid. The different possibilities
examined (not shown) induced modifications on the fluxes of
transaldolase (Tlc and Tlp) and of the cytosolic
triose phosphate to hexose phosphate conversion (TFc) but were
found to have little effect on the other fluxes.
There is ample
evidence that glucose 6-phosphate can be transported into non-green
plastids
(38, 43) . Studies of the route of starch
synthesis using either enzyme activities
(36, 38) or
label distribution (4, 5) indicate that starch glucosyl units arise
from glucose phosphate imported from the cytosol. Our results
essentially confirm this view. However, the high redistribution found
here between the C-1 and C-6 positions in starch glucosyl units
compared with sucrose was not found in other materials. We attributed
this difference to a plastidial transaldolase activity because
relatively high transaldolase and transketolase activities have been
found in amyloplasts of sycamore cells
(46) and plastids of pea
roots
(49) . The alternative hypothesis, resynthesis of hexose
phosphates from triose phosphates, appears less likely because of the
absence, or low activity, of fructose-1,6-bisphosphatase in most
non-green plastids
(36, 37) .
The glycolytic flux of
hexose phosphate to triose phosphates, FT = 0.42, was
1.6 times the flux into the pentose phosphate pathway (P). The
unidirectional flux of triose phosphate production, not including
transaldolase activities, was 0.63, of which 68% was from glycolysis
and 32% from the pentose phosphate pathway. 60% of the triose
phosphates were found to be recycled to hexose phosphates
(TFc). Because of transaldolase activity, triose phosphate
recycling may have been overestimated when calculated from
randomization of C-1 to C-6
(5, 7) . Here, the use of
enrichments allowed this reaction to be distinguished from
transaldolase exchange, resulting in a more accurate value. 28% of the
triose phosphates were found to go to the tricarboxylic acid cycle
(TCA), and the remainder was recycled in the pentose phosphate
pathway or used for the synthesis of fatty acid (Lip).
The
carbon flux to the tricarboxylic acid cycle was found to be divided at
the PEP branch point, with 33% going to PEP carboxylase and 67% to the
pyruvate kinase reaction. Only 8% of pyruvate was found to derive from
C
The similar enrichment of alanine C-3 and
glutamate C-4 () is consistent with the view that
glycolysis is the sole source of acetyl-CoA for the tricarboxylic acid
cycle in glucose-fed tissues. However, since lipids and proteins were
found to be labeled during the long incubation time needed to reach
isotopic steady state (result not shown), this result is not a proof
that the turnover of fatty acids and amino acids provides negligible
amounts of acetyl-CoA to the tricarboxylic acid cycle.
The
contribution of the different pathways to the total flux of CO
The major flux determined in the intermediate
metabolism of the maize root tips was that of synthesis and degradation
of sucrose. This futile cycle has been described recently in a number
of plant tissues
(2, 3, 7, 35) but does
not appear to occur in the more mature portion of the maize
root
(45) . The cost of synthesis and degradation of one sucrose
molecule is 2 ATP, as far as pyrophosphate is used as ATP equivalent
(7). Therefore, 3.1 mol of ATP are consumed in sucrose cycling for each
hexose entering the tissues. The relative flux of ATP regeneration was
calculated from the carbon flux through glycolysis (Glyco = 0.21 produces 0.42 ATP and 0.42 NADH) and the different
steps of pyruvate oxidation in the mitochondria (with CS = 0.12, PEPC = 0.06, and the maximum value
ATP/O = 3). Assuming that the oxidation of cytosolic NADH gives
3 ATP after production of malate, which is then oxidized in the
mitochondria, mitochondrial respiration was calculated to produce a
maximum of 4.5 ATP per hexose molecule entering the root tip. Thus, the
turnover of sucrose consumes 69% of the ATP produced by mitochondrial
respiration. An even higher figure was obtained for banana
tissue
(7) , which is consistent with the absence of biosynthetic
activities linked with growth in that material. This futile cycle could
be involved in the regulation of the concentrations of sucrose and
hexose, which determine the water status of these cells.
The present
work describes intermediate metabolism in growing higher plant tissues
fed with non-limiting amounts of carbohydrate. However, there is
increasing evidence that the supply of carbohydrates fluctuates and
controls the activity of plant cells. Some of the metabolic effects of
sugar starvation have been
described
(17, 18, 19, 20, 21, 22, 23, 24) ,
and it has been established that the expression of some genes is under
metabolic control by sugars or their sugar metabolites
(50) . The
present study provides the basis for a study of the metabolic response
of plant cells to varying carbon availability or other stresses.
Specific enrichments were deduced from the data in
. PEP C-2 (T2), which derives from C-2 or C-5 of the hexose
phosphate, was not labeled and was assumed to have the natural
enrichment, i.e. T2 = 1.1%. Malate C-2 and C-3 are
identical to the C-3 and C-2 of glutamate, respectively, i.e. O2 = O3 = 25.4 ± 0.4%. P2 and P3 are 3 and
34.2 ± 0.5%, respectively. Solving the three equations with
these values gave V
For plastids: 10) St (synthesis of starch); 11)
P (flux of hexose phosphates into the pentose phosphate
pathway). The model indicates that the triose phosphates used by
aldolase (flux P/6) are taken from the triose phosphate pool
and can thus incorporate label in hexose phosphate C-6, in agreement
with the proposal by Viola et al.(6) (see also Ref.
25). 12) A (flux of the fructose phosphates produced from
pentose phosphates, back to the glycolytic hexose phosphate pool). The
model has to account for the loss of enrichments from the cellular
hexose phosphates, for the different redistribution of label in the
hexose units, and for the enrichment of the starch glucosyl C-1 after
incubation with [2-
The enrichment of labeled carbons in the different
compounds are designated by 1 or 2 letters for the compound and a
number, which indicates the carbon position in the molecule: S designates external glucose; Gl designates intracellular
free glucose; H, cytosolic hexose phosphate; G,
starch hexosyl units and plastidial hexose phosphates; T,
triose phosphates; P, pyruvate and alanine; O,
oxalacetate. Glucose influx into root tips was 1.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Equations for cytosolic hexose phosphates are as follows.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Equations for plastidial hexose phosphates are as follows.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Equations for triose phosphates are as follows.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Metabolic steady state equations are as follows.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy TCA = Glyco - Lip. This
equation applies to the PEPC/CS ratio and the
distribution of the PEPC carbon flux to amino acid synthesis described
above.
The rates of
On-line formulae not verified for accuracy where the symbols defined above are used for carbons labeled from
[1-
The enrichments (in %) were
determined from
Maize root tips were incubated
for 12 h with [2-
Relative fluxes were calculated by normalizing rates in hexose units
to the rate of glucose influx (215
nmol
We thank Dr Bryan Collis (School of Biological
Sciences, Bangor, UK) and Ann Collis for improving the English of the
manuscript and Dr Blanc (Université de Bordeaux II, UFR MISS)
for the software DERIVE.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
C or
C. The specific radioactivity of
amino acids and the
C-specific enrichment of specific
carbons of free glucose, sucrose, alanine and glutamate were measured
and used to calculate metabolic fluxes. The non-triose pathways,
including synthesis of polysaccharides, accumulation of free hexoses,
and to a lesser extent starch synthesis, were found to consume 75% of
the glucose entering the root tips. The cycle of synthesis and
hydrolysis of sucrose was found to consume about 70% of the ATP
produced by respiration. The comparison of the specific radioactivities
of amino acids and phospholipid glycerol phosphate after labeling with
[1-
C] or [6-
C]glucose
revealed the operation of the pentose phosphate pathway. The transfer
of label from [2-
C]glucose to carbon-1 of starch
glucosyl units confirmed the operation of this pathway and indicated
that it is located in plastids. It was found to consume 32% of the
hexose phosphates entering the triose pathways. The remaining 68% were
consumed by glycolysis. The determination of the specific enrichment of
carbohydrate carbons -1 and -6 after labeling with
[1-
C]glucose indicated that both the conversion
of triose phosphates back to hexose phosphates and the transaldolase
exchange contributed to this randomization. Of the triose phosphates
produced by glycolysis and the pentose phosphate pathway, about 60%
were found to be recycled to hexose phosphates, and 28% were directed
to the tricarboxylic acid cycle. Of this 28%, two-thirds were found to
be directed through the pyruvate kinase branch and one-third through
the phosphoenolpyruvate branch. The latter essentially has an
anaplerotic function since little malate was found to be converted to
pyruvate (malic enzyme reaction).
(
)
branch point and the
malic enzyme flux
(10) , were determined in germinating embryos
of lettuce. However, the situation in this material is unusual since
fatty acids rather than carbohydrates are the source of acetyl-CoA
entering the citrate synthase reaction
(10) ; therefore, little
is known of the relationship between glycolysis and the tricarboxylic
acid cycle in the glucose-metabolizing tissue, which is the common
situation in plants. Although most of the reactions occurring in plant
intermediate metabolism have now been identified, our knowledge is
still limited by the lack of quantitative determination of fluxes in
extended networks.
CO
production from
glucose labeled on C-1 or C-6, Gibbs and Beevers
(11) detected
no pentose phosphate pathway. As a result, it is sometimes assumed that
the metabolism of the root tips of maize is very simple. The
calculation of the rate of aerobic glycolysis from CO
evolution presented by Hole et al.(12) requires
CO
to be produced after glycolysis only, as suggested in
Ref. 11, and to be the only product of glucose oxidation. However, the
pentose phosphate pathway is generally considered to account for
10-15% of the oxidation of carbohydrates in plant tissues (13).
Since its main role is the supply of NADPH for biosynthesis, this
pathway would be expected to be particularly active in the root tip,
which is essentially a meristematic tissue. In aerobic root tips, the
shortage of carbohydrate rapidly induces dramatic changes in metabolic
activities, with a decrease in the rate of respiration
(17) , a
change in the carbon source of respiration from carbohydrates to lipids
and proteins
(18, 19) with an increase in the
peroxisomal and mitochondrial
-oxidation
activities
(20, 21) , and the induction of proteolytic
enzymes
(22) . Similar changes have been described in other plant
material such as sycamore cells in culture
(23) or asparagus
spears (24). However, our knowledge of the metabolic network affected
by these changes is incomplete and lacks quantitative data, thus
precluding any prediction of their consequences and any comprehension
of the mechanisms leading to cell acclimation, or failure, under stress
conditions.
C]- or
[
C]- labeled glucose and used metabolic pathway
modeling to describe isotope distribution. This method has been applied
successfully to animals (25-27) but has rarely been used for
plants, and with monocompartmental models
only
(5, 7, 10) . We used NMR to determine the
C enrichment of specific carbons of carbohydrates and
amino acids. Using these data, we provide estimates for 20 metabolic
fluxes, from sucrose turnover to inputs into the tricarboxylic acid
cycle. We show definitely that the pentose phosphate pathway is active
in maize root tips and that it is compartmented in the plastid; we
suggest that the synthesis of pentans may account for identical
CO
evolution from [1-
C]
or [6-
C]glucose. The use of carbon enrichments
provided evidence for a contribution of the transaldolase reaction to
the randomization of hexose phosphate carbons, which would not be
detected from label exchange ratios. The model provided can be used to
calculate fluxes from labeling data in intermediary metabolism in other
plant systems.
Materials
Maize seeds (Z. mays L. cv. DEA, Pioneer France Mas, France) were germinated for 3 days
in the dark at 25 °C as described
(18) . The 3-mm tips of
primary roots were excised. Before labeling, they were starved of
carbohydrates for 4 h in the medium described in Ref. 18 called
``medium A.'' This treatment depletes starch and fructose and
decreases the pools of sucrose and glucose to 20 and 50% of their
initial values, respectively
(17, 18) . These tips,
called ``prestarved root tips,'' were then transferred to
medium A supplemented with 200 mM labeled glucose. This
concentration of glucose is necessary to maintain their respiration
rate
(17) . For C labeling, triplicate samples of 30
prestarved root tips were incubated in 5-ml syringes containing 2 ml of
medium A with 200 mM [1-
C],
[6-
C], or [2-
C]glucose
(0.25-0.5 MBq/mmol) and bubbled with a N
/O
mixture (50/50, v/v). For NMR experiments, 500 or 1000 prestarved
maize root tips were incubated in medium A (50 ml/1000 tips),
supplemented with 200 mM [1-
C]glucose.
After incubation, the root tips were washed with abundant ice-cold
water as described
(28) to eliminate exogenous glucose and then
frozen in liquid nitrogen. The
CO
produced was
trapped by bubbling through a series of three tubes containing 2 ml of
2% KOH. At the end of incubation, the radioactivity of the KOH
solutions was counted in a Packard scintillation counter.
Chemicals
Analytical-grade mineral salts
and amyloglucosidase were purchased from Merck (Darmstadt, Germany),
and HPLC grade solvents were from Prolabo (Paris).
[1-C]Glucose (1.856 GBq/mmol),
[2-
C]glucose (1.7 GBq/mmol), and
[6-
C]glucose (0.263 GBq/mmol) were purchased
from NEN (Paris) or Dositech (Paris), and
[1-
C]glucose (99% enrichment) was purchased from
Commissariat l'Energie Atomique (Gif-sur-Yvette, France). Yeast
hexokinase, Leuconostoc glucose-6-phosphate dehydrogenase, and
Torula yeast 6-phosphogluconate dehydrogenase were obtained from Sigma.
Analysis of Metabolites
The extraction of
soluble components was performed using boiling aqueous solutions of
ethanol, as previously described
(10) . The extract was
concentrated by evaporation; water was added to make a volume of 1 or 2
ml, and this total extract was used for the preparation of lipids and
water-soluble compounds. Lipids were extracted and saponified, and the
fatty acids were extracted as described
(29) . The radioactivity
remaining in the ethanol-water phase was identified as glycerol
phosphate by passing the ethanol-water phase through an anion exchange
resin (Dowex 1-X8, Bio-Rad, formate). The amino acid fraction was
separated by ion exchange chromatography using standard procedures
(10) and analyzed by HPLC after derivatization with
o-phthaldialdehyde as described
(19) ; the specific
radioactivity of each amino acid was determined as in Ref. 10. For HPLC
analysis of sugars, the water-soluble extract was deionized using anion
and cation exchange resins and analyzed as described
(30) on a
Aminex HPX-87C column (Bio-Rad). Each peak was collected and counted
for determination of the specific radioactivity. The residue of
ethanolic extraction was used for the analysis of starch. It was washed
successively with 20% ethanol and water (1.5 ml each for 30 roots), and
starch was converted to glucose and analyzed by HPLC as
described
(30) . Total protein was extracted and determined after
mineralization as described
(19) . For NMR analysis, glutamate
was separated from the amino acid fraction as in Ref. 10, passed
through a Chelex 100 column, dried, and dissolved in 350 µl of
DO. The fraction containing the amino acids minus glutamate
and aspartate was used for the
H NMR analysis of alanine.
,
6.5 mM cysteine, 3 units of hexokinase, 5 units of
glucose-6-phosphate dehydrogenase (in 20 µl of 0.2 M
MgSO
/glycerol (50/50, w/w)), and 3 units of
6-phosphogluconate dehydrogenase. The center well contained a filter
paper with 0.2 ml of 2% (w/v) KOH for CO
collection. The
reaction was started by adding an extract volume containing 400 nmol of
glucose, and the vial was stoppered immediately. After 2 h, 200 µl
of 1 M HCl, kept in the sidearm of the Warburg vial, was mixed
with the reaction medium to liberate CO
. After 30 min, the
filter paper was recovered, and the center well was rinsed three times
with 200 µl of water; 500 µl of this solution was counted. The
yield of the decarboxylation reaction, determined with standard
[1-
C]glucose, was 82 ± 2%.
NMR Spectroscopy
Extracts for NMR
spectroscopy analysis were evaporated to dryness under vacuum (Speed
Vac) and dissolved in 350 µl of DO.
C NMR
spectra were obtained at 100.6 MHz with a Bruker AM 400 spectrometer
with a 5-mm reverse probe. Spectra were collected using the composite
proton decoupling sequence, a 45° flip angle, a 4-s interpulse
delay, and 16 K points.
H NMR spectra were obtained at 400
MHz, using a 45° flip angle, a 6-s interpulse delay, and 4 K
points. Except for the analysis of sugar fractions, water resonance was
eliminated using a water presaturation sequence. Prior to Fourier
transformation,
C-free induction decays were zero-filled
to improve digital resolution and multiplied by an exponential function
to improve the signal-to-noise ratio (1.5 Hz line broadening). Peak
assignment was done according to Refs. 32 and 33 and from spectra of
pure compounds.
C enrichment of glutamate
carbons C-2, C-3, and C-4, of alanine C-3, of
and
glucose
C-1, and of sucrose glucosyl C-1 were determined from
H NMR
spectra, as the ratio of the area of the satellites
(
C-
H coupling) to the total area of the
multiplet. Deconvolution of the signals was used when necessary, as in
Fig. 5
and Fig. 6. The relative enrichments of the glucose
and sucrose carbons C-2 to C-6 were determined from
C
spectra, from the absolute enrichment of C-1 determined by
H NMR, and the comparison of their peak areas to that of
the same resonances from non-enriched sugars, the latter being equally
labeled at all carbons by the 1.1% natural abundance. This method
avoids the errors due to nuclear Overhauser and relaxation effects,
which would occur in the determination of relative enrichments by
direct comparison of peak areas in
C spectra. The same
method was used to determine the enrichment of alanine C-2 from the
absolute enrichment of alanine C-3 and to confirm the enrichments of
the glutamate carbons.
Figure 5:
C and
H
(inset) NMR spectra of glucose from starch hydrolysate.
Pre-starved maize root tips were incubated for 12 h with
[1-
C]glucose. Starch was extracted and
hydrolyzed to glucose as described under ``Experimental
Procedures.''
H and
C spectra represent
the accumulation of 250 and 1000 scans, respectively. Gi
and Gi
indicate the resonance of carbon i of
and
glucose, respectively. E is the
C
enrichment of
glucose C-1, expressed in
%.
Figure 6:
C (top) and
H (bottom) spectra of purified glutamate after 12
h of labeling with [1-
C]glucose. Pre-starved
maize root tips were incubated for 12 h with 200 mM
[1-
C]glucose. Glutamate was purified as
described under ``Experimental Procedures.''
H
and
C spectra represent the accumulation of 128 and 10,000
scans, respectively. E is
C enrichment expressed
in %.
Calculations
The resolution of
simultaneous algebraic equations was done using the solver DERIVE (Soft
Warehouse, Inc.).
Establishment of Isotopic and Metabolic Steady
State Upon Incubation with Glucose
The time needed to reach isotopic and metabolic steady state
was determined by following the evolution of
CO
, using [1-
C]glucose
as substrate. At isotopic steady state, specific radioactivities of
intermediates of glycolysis and the tricarboxylic acid cycle are
constant. Therefore, the rate of labeled CO
evolution
becomes constant. When root tips were not ``prestarved,'' the
rate of labeled CO
evolution increased continuously for
more than 16 h, thus indicating that a longer incubation would be
needed to reach steady state. When the endogenous pool of carbohydrates
was depleted by a starvation pretreatment, the rate of labeled CO
evolution became constant after 10 h (data not shown). The
specific radioactivities of sucrose and fructose were already steady
within 2 h (Fig. 1). At the same time, that of intracellular
glucose was only about 60% of the final value; it then increased slowly
to reach the steady-state value after 10 h. At that time, intracellular
glucose and fructose had the same specific radioactivity, which was 90%
that of the tracer glucose. The specific radioactivity of sucrose was
twice that of glucose or fructose (Fig. 1). In
C
labeling experiments, an incubation time of 12 h was chosen. We
verified on the NMR spectra that the enrichments of carbohydrates and
of the amino acids glutamate and aspartate were the same at 12 and 18 h
of incubation with [1-
C]glucose, thus confirming
the establishment of steady state at 12 h.
Figure 1:
Specific radioactivities of glucose
(), fructose (
), and sucrose (
) during the
incubation of maize root tips with [1-
C]glucose.
Excised maize root tips were pre-incubated for 4 h in the buffered
mineral solution described under ``Experimental Procedures''
in the absence of glucose (prestarved tips) and then incubated with 200
mM [1-
C]glucose (23.2
dpm
nmol
). The specific radioactivities of
soluble sugars were determined after HPLC analysis as described under
``Experimental Procedures.'' Data presented are the mean of
determinations on two samples in a representative experiment. Range was
±5% for glucose and fructose and ±10% for sucrose.
Qualitatively similar data were obtained in four experiments with
[1-
C]glucose at different specific
radioactivities.
Root tip growth continued
during incubation in the presence of glucose. Average length increased
from 3 to 10 mm, and average dry weight increased from 0.35 to 0.81
mg/tip in 12 h.
Setting Up the Metabolic Scheme
COand Amino Acid Labeling with
[1-
C] or
[6-
C]Glucose as Evidence for an Activity of
the Pentose Phosphate Pathway-The lower enrichment of
endogenous carbohydrates, compared with the precursor, might be
explained by the loss of
CO
through the
pentose phosphate pathway activity, the presence of an inactive pool of
carbohydrates, and the gluconeogenesis of unlabeled carbohydrates from
endogenous precursors. The hypothesis of the pentose phosphate pathway
activity was tested by incubations with
[1-
C]glucose and
[6-
C]glucose. In agreement with earlier
results
(11) , no difference in the production of labeled
CO
was found between the two tracers (data not shown),
indicating no pentose phosphate activity. However, as shown in
Fig. 2 , the specific radioactivity of amino acids was
22-30% higher with [6-
C]glucose than with
[1-
C]glucose. In the absence of the pentose
phosphate pathway, this difference could be explained by the
non-equilibrium of the triose phosphate isomerase reaction and the
output of dihydroxyacetone phosphate (labeled with glucose C-1) to
glycerol phosphate for lipid synthesis. In this case, the glycerol
moiety of lipids would contain more label from
[1-
C] glucose than
[6-
C]glucose. However, the rate of incorporation
of [1-
C]glucose into lipid glycerol phosphate
was only 70% of that of [6-
C]glucose
(Fig. 2D). This indicates that the specific
radioactivity of dihydroxyacetone phosphate was 30% lower for
[1-
C]glucose than for
[6-
C]glucose. The observed similar differences
in the specific radioactivities of dihydroxyacetone phosphate and
alanine with [1-
C]glucose or
[6-
C]glucose further indicated that the triose
phosphate isomerase reaction was close to equilibrium. This disproves
the hypothesis of non-equilibrium of the triose phosphates and favors
the hypothesis of a flux through the oxidative branch of the pentose
phosphate pathway. To account for the absence of difference in the
production of labeled CO
from both tracers, we assumed
extra production of
CO
from
[6-
C]glucose in the decarboxylation of
UDP-glucuronic acid to UDP-xylose for pentan synthesis; this non-triose
pathway of carbohydrate metabolism is particularly active in elongating
plant cells
(13, 34) .
Figure 2:
Specific radioactivities of amino acids
and production of labeled glycerol phosphate during incubation of maize
root tips with [1-C]glucose (
) or
[6-
C]glucose (
). Prestarved maize root tips
were incubated with 200 mM [1-
C] or
[6-
C]glucose (30 ± 0.6 dpm/nmol).
Specific radioactivities of glutamate (A), aspartate
(B), and alanine (C) were determined after HPLC
analysis. Glycerol phosphate (D) was purified as described
under ``Experimental Procedures,'' and the incorporated
radioactivity was counted. Data are means (±S.D.) of
determinations on four samples from two independent experiments for the
amino acids and on two samples for glycerol
phosphate.
Labeling of Carbohydrates by Sucrose Turnover and Cycling
between Hexose Phosphates and Triose Phosphates
Fig. 3
shows a typical C NMR spectrum of the
ethanolic extract of maize root tips after 12 h of incubation with
[1-
C]glucose. Resonances assigned to glucose,
sucrose, and fructose carbons are clearly visible. The spectrum shows
numerous peaks of labeled intermediates related to the tricarboxylic
acid cycle, including the amino acids glutamate, glutamine, aspartate,
and alanine and the carboxylic acids citrate and malate. The highest
amounts of
C are at C-1 of glucose and C-1 of the glucosyl
and fructosyl moieties of sucrose. The spectrum also shows appreciably
more labeling at C-6 than at the other carbons of glucose and the
sucrose moieties.
Figure 3:
Proton-decoupled C NMR
spectrum of the ethanolic extract of maize root tips after 12 h of
labeling with [1-
C]glucose. The spectrum
represents the accumulation of 3,200 scans. InsetA,
spectrum region (14-57 ppm) displaying amino acid resonances;
B, spectrum region (66-83 ppm) displaying sugar
resonances. Peak assignments: Mal, malate; Cit,
citrate; Gi
and Gi
, Si and
Si, resonances of carbon number i of
and
glucose
and of the fructosyl and glucosyl moieties of sucrose, respectively;
D, E, Q, and A indicate the
resonances of carbon i of aspartate, glutamate, glutamine, and alanine,
respectively.
We verified that the glucose present in the medium
at the end of incubation was identical to the glucose supplied. Thus,
glucose entering the maize root tips is labeled to 99% on C-1 and not
labeled on the other carbons, which means that their C
content is the natural enrichment, 1.1%. The enrichments of carbons of
interest in selected metabolites are shown in . The label
distributions observed in the glucosyl and fructosyl moieties of
sucrose were similar, with averaged enrichments of 81 ± 1% and
14.5 ± 1.5% on C-1 and C-6, respectively (). Sucrose
is a cytosolic compound that can be labeled on its glucosyl and
fructosyl moieties when synthesized through the sucrose phosphate
synthase reaction
(35) . The substrates for this reaction come
from the cytosolic glucose phosphate and fructose phosphate pools.
Given this, the cytosolic hexose phosphates can be considered as a
single pool where the C-1 or C-6 enrichments are the average enrichment
of the corresponding carbons in the hexosyl moieties of sucrose
(). The labeling of free glucose on C-6 suggests that
intracellular glucose is formed from hexose phosphates, presumably via
their incorporation into sucrose and subsequent hydrolysis of sucrose
by an invertase
(1, 2) , which produces free glucose and
fructose. A
C NMR spectrum of purified free fructose
showed that its C-1 and C-6 were labeled (not shown), which is
consistent with sucrose cycling through the sucrose phosphate synthase
and invertase reactions. However, the hydrolysis of hexose phosphates
by a phosphatase would have a similar effect to, and cannot be
distinguished from, the sucrose phosphate synthase/invertase pathway of
sucrose turnover. Intracellular free glucose showed less randomization
than the hexosyl units of sucrose (), which is consistent
with intracellular glucose being a mixture of entering precursor and
recycled sucrose-glucosyl. The presence of residual extracellular
glucose would increase this difference but has previously been shown to
be negligible
(28) .
Figure 4:
Pathways of carbohydrate metabolism in
glucose-fed maize root tips. Metabolic pathways have been identified
using the labeling of intermediates described in the text. Flux names
are as defined under ``Appendix.'' Flux values are given in
Table III.
The glucose carbons C-3 and C-4 were found to be
slightly enriched to about 1 or 2% above natural enrichment (not
shown), which suggested some resynthesis of pentose phosphates from
fructose phosphate and triose phosphate through the non-oxidative
branch of the pathway. This would imply that not only the transaldolase
but also the transketolase reaction is reversible
(9) . However,
the very low enrichment of these carbons indicated that this flux would
be a minor one. Since the reversal of this reaction was found to have
little effect on the calculation of fluxes in glycolysis and the
pentose phosphate pathway
(9) , it was not included in the
metabolic scheme. The triose phosphates used in the resynthesis of
hexose phosphates might also be formed by gluconeogenesis from organic
acids. In addition to glucose C-6, gluconeogenesis would also label
glucose C-2 and C-5 from the labeled C-2 of oxalacetate (see below).
The absence of any significant label on glucose and sucrose C-2 and C-5
(Fig. 3) indicated the absence of gluconeogenesis.
Plastidial Hexose Phosphates Import from the Cytosol,
and Cycling in the Transaldolase Reaction
Starch was
studied by H and
C NMR after conversion to
glucose. The enrichments of starch glucosyl C-1 and C-6 were 65 and
18-19%, respectively (Fig. 5, ). If starch were
formed from triose phosphates, the enrichments of the C-1 and C-6 of
its glucosyl units would be similar
(4) . The higher enrichment
of C-1, similar to that observed in other non-green
plastids
(4, 5, 6) , indicated that starch was
formed essentially from hexose phosphates imported into the plastids.
However, in the present case, we found more randomization in the
glucosyl units of starch than in glucose or the hexosyl units of
sucrose. Therefore, an exchange through the transaldolase reaction in
the plastid, similar to that in the cytosol, was introduced into the
metabolic scheme. Recycling of triose phosphates to hexose phosphates
could account for this effect but was not included because recent
evidence from enzyme studies indicates that fructose-1,6-bisphosphatase
activity is absent
(36) or has a low activity
(37) in
plastids of most starch-storing tissues. The different labeling of
hexosyl units in the cytosol and in the plastids suggests that there is
no significant efflux of hexose phosphates from the plastids to the
cytosol. Labeling with [2-
C]Glucose and
Localization of the Pentose Phosphate Pathway-To obtain
additional evidence for the occurrence of the pentose phosphate
pathway, maize root tips were labeled with
[2-
C]glucose. A flux through this pathway, with
recycling of triose phosphates to fructose 6-phosphate, would lead to
the labeling of hexose phosphate C-1, which can be detected by the
analysis of glucose, sucrose, or starch
(6) . Free glucose and
glucose produced from starch hydrolysis were purified, and the specific
radioactivity of their C-1 was determined by decarboxylation. The
specific radioactivity of glucose C-1 was only 0.38
dpm
nmol
compared to 1.65 ± 0.01 for
starch glucosyl units; these values are equivalent to
C
enrichments of 2.6 and 11.1%, respectively (). The
labeling of C-1 of either glucose or starch glucosyl confirmed the
activity of the pentose phosphate pathway, and the higher labeling in
starch than glucose indicated that this pathway was located essentially
in the plastids. In fact, simulations using the metabolic model (see
below) indicated that the labeling of cytosolic glucose C-1 could be
accounted for by the reversibility of glycolysis from triose
phosphates; therefore, cytosolic pentose phosphate pathway was not
included in the metabolic scheme (Fig. 4).
Carbon Fluxes through the Tricarboxylic Acid
Cycle
In animal and bacterial cells, alanine, glutamate,
and aspartate are usually considered to be in equilibrium with the
-oxoacids pyruvate,
-oxoglutarate, and oxalacetate,
respectively (39). This appears to be true in plant cells, where the
labeling of these amino acids was found to reflect that of the
corresponding organic acids
(10) . We considered that, in maize
root tips, where transaminase activities are high
(19) , the
C distribution in these amino acids also reflects the
distribution in the corresponding
-oxoacids. shows
the
C enrichment of the non-carboxylic carbons of these
amino acids, as determined using
H NMR (Fig. 6).
C enrichment of alanine C-3 was 34.2%, and that of
alanine C-2 was 3%. Glycolysis introduces the C-1 or C-6 of glucose
into alanine C-3. In the absence of a diluting flux between hexose
phosphates and pyruvate, the enrichment of alanine C-3 would be equal
to the average of the hexose phosphate C-1 and C-6 enrichments
(), i.e. (81 + 14.5)/2 = 48%. The
dilution of alanine C-3 from 48 to 34.2% further confirms the activity
of the pentose phosphate pathway. Alanine C-2 could be labeled from a
labeled C
organic acid, oxalacetate or malate, through the
malic enzyme reaction or through the PEP carboxykinase and pyruvate
kinase reactions. As previously observed
(10) , these two
pathways cannot be distinguished from each other using the present
method. However, since PEP carboxykinase activity is low in maize root
tips
(16) , this flux was attributed to malic enzyme. Plant
mitochondria differ from animal mitochondria in containing a malic
enzyme activity, which converts malate to pyruvate
(40) . Since
the enrichment of alanine C-2 indicates that such an activity operates
in the maize root tips, it was included in the model.
C enrichments of glutamate C-2 and
glutamate C-3 () were identical to each other and close to
25.5%; this value was lower than that for glutamate C-4, indicating the
entry of a diluting flux of C
compounds into the
tricarboxylic acid cycle
(26) . In glucose-fed tissues, PEP
carboxylase activity is the most likely source of anaplerotic
carbon
(13, 41) . This reaction provides oxalacetate
molecules labeled on C-3; label can be transferred to the C-2 position
by the fumarase reaction. These carbons then provide the glutamate
carbons C-2 and C-3. In the present case, the identical
C
enrichment of glutamate C-2 to glutamate C-3 indicated that the
fumarase reaction was close to equilibrium. In addition, it also
indicated the absence of the form of channelling, which converts
oxoglutarate C-4 to oxalacetate C-3, which would give a higher labeling
of glutamate C-2 than C-3
(27) ; however, the alternative form of
channelling, which would convert oxoglutarate C-4 to oxalacetate C-2,
could not be excluded because, in this case, the carbons 2 and 3 of
glutamate would eventually become equally labeled (42).
Determination of Fluxes
The metabolic network is shown in Fig. 4, and flux
parameters are defined under ``Appendix'' and shown in
I. V(G) (the rate of glucose uptake), SS (the flux to soluble sugar accumulation), St (the rate of
starch accumulation), Lip (the rate of fatty acid synthesis),
and V(CO) (the rate of CO
evolution) were determined directly (I,
Fig. 7 ).
Figure 7:
Changes
in soluble sugars (glucose (), sucrose (
), and fructose
(
)) in excised maize root tips. Maize root tips were pre-starved
for 4 h and, at the time indicated by an arrow, tranferred to
an incubation medium containing 200 mM glucose. The data are
the mean of determinations on four samples from two independent
experiments.
Other fluxes of glucose metabolism were calculated
using mathematical models of tracer distribution at metabolic and
isotopic steady states for the metabolic network shown in Fig. 4.
Equations were written according to Katz and Rognstad
(9) . The
flux variables and equations are presented under
``Appendix.'' The relative fluxes have been determined in
three steps as described under ``Appendix'': (i) estimation
of the activity of ``malic enzyme'' relative to that of PEP
carboxylase (V) and determination of the
enrichment of PEP C-3 (T3), (ii) estimation of the carbon fluxes into
the tricarboxylic acid cycle through the PEPC and citrate synthase
reactions relative to each other (V
and
V
), and (iii) estimation of the fluxes of sugar
metabolism. Flux values are given in I.
Malic Enzyme Flux
As shown in Fig. 4,
pyruvate is formed either from PEP by pyruvate kinase or from malate by
malic enzyme. A model limited to this set of three compounds was used
to calculate both V relative to
V
+ V
and T3, as
described under ``Appendix.'' The value of V
= 0.08 indicated that the malic enzyme flux only provided
8% of the pyruvate entering the tricarboxylic acid cycle. The effect of
the malic enzyme flux was to enrich pyruvate C-2 (P2) and dilute
pyruvate C-3 (P3) relative to the triose phosphate carbons T2 and T3.
The value of T2 was the natural enrichment 1.1%; that of T3 was 35.0
± 0.6%, i.e. not very different from P3 (34.2 ±
0.5%, ). The value of V
was
normalized to V(G), using the value of TCA, the flux
entering the tricarboxylic acid cycle (see below), giving Mal = 0.01 (I).
PEP Carboxylase and Citrate Synthase
Fluxes
In the absence of the PEPC flux, the enrichment of
the glutamate carbons C-2 and C-3 would be equal to that of glutamate
C-4
(25) , i.e. 33.8 ± 0.6% (); the
observed values, however, are 25.4 ± 0.4% (), thus
indicating the operation of the PEP carboxylase reaction. This reaction
produces oxalacetate whose C-3 is T3, enriched to 35 ± 0.6% (see
above), and C-2 is T2 (1.1%). After randomization through the fumarase
reaction, the mean enrichment of these oxalacetate carbons is 18%. This
flux mixes with that from the tricarboxylic acid cycle, giving
oxalacetate carbons O2 and O3 identical to the glutamate carbons C-3
and C-2. The ratio of the carbon flux through PEP carboxylase to that
through citrate synthase was calculated from these values, according to
Ref. 10 (see ``Appendix''), giving
V/V
= 1.03
± 0.14. This value indicates that 52 ± 7% of the carbon
entered the tricarboxylic acid cycle as oxalacetate and 48% as
acetyl-CoA. Since one triose phosphate molecule brings four carbons
when entering the tricarboxylic acid cycle through the PEPC reaction
and only two carbons through the citrate synthase reaction, it was
calculated that, of the triose phosphates entering the tricarboxylic
acid cycle, 35 ± 5% went through the PEPC reaction and 65%
through the citrate synthase reaction. The corresponding values
normalized to V(G) were PEPC = 0.06 and CS = 0.12 (I).
C spectra
(Fig. 6) by the method of Malloy et al.(26) was
in agreement with our direct determination from the
H NMR
spectrum. However, fluxes entering the tricarboxylic acid cycle could
not be determined reliably because, in the range of low enrichments
obtained in the present experiment, small errors in the relative size
of doublets and triplets of glutamate resulted in large errors on
calculated carbon fluxes (results not shown).
Glycolysis and the Pentose Phosphate
Pathway
The equations describing the labeling of free
glucose, cytosolic and plastidial hexose phosphates, and triose
phosphates at isotopic steady state are given under
``Appendix.'' In the case of
[1-C]glucose labeling, only the equations
describing the hexose C-1 and C-6 and the triose phosphate C-3 were
used.
Figure 8:
Graphic determination of the rate of
sucrose synthesis and degradation (Suc). Graphic
representation of Equation 1, Suc H1 + S1 = (1
+ Suc) Gl1 (``Appendix''), shows Gl1 as a
function of Suc for the two extreme values of the enrichment
of cytosolic hexose phosphate C-1 (H1 = 0.79 and 0.81). Graphic
representation of Equation 6, Suc H6 + S6 = (1
+ Suc) Gl6, shows Gl6 as a function of Suc for
the two extreme values of the enrichment of cytosolic hexose phosphate
C-6 (H6 = 0.13 and 0.14). For each equation, an area was
delimited to take into account the variation of Gl1 from 0.83 and 0.84
and that of Gl6 from 0.09 and 0.1. The enrichment values used here were
taken from Table I and corrected for the 1.1% natural abundance. The
overlap region corresponds to the interval of variation of
Suc, 2.8-3.4.
The Equations 13, 18, 21, 23, 25, and 26 were then
solved simultaneously to determine the relative fluxes Pl,
P, A, Tlp, Pol, and Pent. The
results (I) indicate that 22 ± 2% of the glucose
entering the cell goes to plastids (flux Pl). These hexose
phosphates are essentially metabolized through the pentose phosphate
pathway (p = 0.27 ± 0.03) and converted to
triose phosphates; the flux of fructose phosphate found to be recycled
to glucose phosphate was A = 0.06 ± 0.02, which
is 22% of the flux entering the pentose phosphate pathway. Most of the
triose phosphates produced in plastids were found to be exported to the
cytosol. A small part may remain in the plastid and be oxidized by the
plastidial pyruvate dehydrogenase, thus contributing to fatty acid
synthesis
(43) . The relative glycolytic fluxes from hexose
phosphates to triose phosphates, FT, and from triose
phosphates to PEP, Glyco, were estimated to be 0.42 and 0.21,
respectively. The plastidial transaldolase flux was calculated with a
large error (Tlp = 0.04 ± 0.04). Absolute values
of fluxes (I) were calculated using the relative values
and the rate of glucose influx into maize root tips (V(G) = 215
nmolh
tip
).
Validation of the Model
To validate the
model, the general equations given in Annex were used to calculate the
expected C-1 enrichments of free glucose and the plastidial hexose
phosphates after labeling with [2-C]glucose,
using the flux values of I and C-2 enrichment equal to 98%
above natural abundance. The calculated enrichments of free glucose C-1
and starch glucosyl C-1 were Gl1 = 2.5 ± 0.5% and G1
= 15.3 ± 2.5%, respectively, to be compared with the
experimental values 2.6 ± 0.2% and 11.1 ± 0.1%
(). Since G1 is very sensitive to the flux values A and Pl, which were determined with large relative errors,
we conclude that the model correctly accounts for the experimental
data.
h
tip
,
would be equivalent to a weight increase of 19
µg
h
tip
. The
residue of the ethanol/water extraction includes cellulose, which
represents 47% of the structural carbohydrate in young maize
plants
(44) ; the increase in the dry weight of this residue was
found to be 8
µg
h
tip
. The
values of Pent and Pol calculated by the model are
therefore consistent with this experimental value. The PEPC flux of 13
nmol
h
tip
would
provide carbon for the synthesis of 26
nmol
h
tip
of amino
acids of the aspartate and glutamate families; other amino acids do not
require this pathway. The measured increase in total protein, 6
± 2
µg
h
tip
,
corresponds to about 60 ± 20
nmol
h
tip
of amino
acids, which also is in reasonable agreement with the estimation of
PEPC. The relative net rate of CO
evolution
calculated from Equation 25 and the mean value of fluxes shown in
I is 0.182, which is close to the experimental value,
V(CO
) = 0.18.
C]glucose and
H and
C NMR analysis of extracts for determination of
C enrichments. Additional results were obtained using
[
C]glucose labeled on specific carbons. The
metabolism of glucose studied in the present work is likely to reflect
the metabolism of carbohydrates in this organ in vivo for the
following reasons. (i) The concentration of glucose used here (200
mM) appears to be close to that seen by the root tip cells
since it is intermediate between the sugar concentration in the phloem,
about 0.5 M(34) , and that in the root tip cells, about
80 mM(17, 18) ; moreover, it has been shown
that this glucose concentration is necessary to sustain the normal
respiration rate of the root tips
(17) and to avoid the increase
in proteolytic activities, which results from limited sugar
supply
(22) . (ii) Glucose is the major carbohydrate in root tip
cells
(17, 18) . (iii) Although the carbon starvation
pretreatment used here may appear artificial, it probably mimics a
situation that commonly occurs in normal plant life (Refs. 18 and 20
and references therein). (iv) Finally, under the conditions used, the
excised root tips continued growing at a high rate.
from [1-
C] or
[6-
C]glucose (Ref. 11 and this study) despite
the high activity of the pentose phosphate pathway found in the present
work. It has been pointed out that pentan synthesis might affect the
interpretation of CO
labeling data
(13) , but the
flux in this pathway had not been determined before. Since a high
activity of pentan synthesis would be expected in a growing tissue for
cell wall formation, it was included in the model to balance the
production of labeled CO
by the pentose phosphate pathway.
This non-triose pathway, flux Pent, was found to consume 19%
of the glucose entering the root tip cells.
C]glucose to the C-1 position of starch
glucosyl. Indeed, a simulation of the latter experiment indicated that
the cytosolic resynthesis of hexose phosphates from triose phosphates
(TFc) was sufficient to account for the labeling of cytosolic
glucose C-1 from [2-
C]glucose, thus leaving no
evidence for any significant activity of the oxidative pentose
phosphate pathway in the cytosol. The oxidative pentose phosphate
pathway (P) metabolized 27% of the glucose entering the maize
root tips, and 38% of the hexose phosphates metabolized in triose
phosphate pathways. A similarly high activity of the oxidative pentose
phosphate pathway was found in cells of Chenopodium
rubrum(5) . In isolated pea chloroplasts, the activities of
the two dehydrogenases of the pentose phosphate pathway were found to
be in excess of the flux of glucose consumption by
respiration
(48) .
compounds, thus suggesting that most of the PEP
carboxylase flux was used for biosynthesis from tricarboxylic acid
cycle intermediates, presumably the biosynthesis of amino acids of the
aspartate and glutamate families. In another quantitative study of the
malic enzyme flux
(10) , it was also found that this flux was a
minor one. It might become higher when C
compounds, such as
malate or asparagine, are being used as respiratory
substrates
(19) .
was calculated from Equation 25 (``Appendix'') and flux
values from I. The tricarboxylic acid cycle, including the
pyruvate dehydrogenase reaction, contributed 53%, the pentose phosphate
pathway 24%, and pentan synthesis 17% of total CO
evolved.
Thus, the estimation of the rate of glycolysis from the rate of
CO
evolution would overestimate the pyruvate kinase flux by
about 50% (compare V(CO
) = 0.18
and CS = 0.12) but would underestimate the fructose
phosphate to triose phosphate flux by a factor of 2.3 (compare
V(CO
) with FT = 0.42 in
I).
Malic Enzyme Flux and PEP C-3
Enrichment
Pyruvate is formed either from PEP by pyruvate
kinase (flux V) or from malate by malic enzyme
(flux V
) and is degraded to acetyl-CoA by
pyruvate dehydrogenase; pyruvate outflow to alanine for protein
synthesis has been neglected. A sub-model limited to this set of
reactions was used. The sum of pyruvate formation fluxes was normalized
to 1 so that at metabolic steady state, V
+
V
= 1. T2 and T3, O2 and O3, and P2 and
P3 represent the enrichments of carbons 2 and 3 of PEP, oxalacetate,
and pyruvate (or alanine), respectively. At isotopic steady state,
V
.T2 + V
.O2 =
P2 and V
.T3 + V
.O3
= P3.
= 0.08 and T3
= 35 ± 0.6%. V
was then normalized
to V(G) to give Mal = 0.01 as described in the
text (I).
PEPC and CS Reactions
The relative fluxes
through the PEPC (PEPC) and the citrate synthase (CS)
reactions were determined from the corresponding values
V and V
, calculated using
the model of Salon et al.(10) , which uses specific
enrichments to estimate the carbons fluxes entering the tricarboxylic
acid cycle. The flux parameters were set as follows. (i) Since no
isocitrate lyase activity
(20) and no gluconeogenesis (this
article) were detected, carbon input from the glyoxylic cycle and
output to gluconeogenesis were set to 0. (ii) The only source of
acetyl-CoA was pyruvate (i.e. no contribution from fatty
acids). (iii) In the absence of an experimental value, we assumed that
the distribution of the anaplerotic oxalacetate flux resulting from the
PEPC activity was divided equally to glutamate- and aspartate-derived
amino acids; this corresponds to carbon fluxes of 45% to aspartate and
55% to glutamate. (iv) To account for the complete randomization of
oxalacetate C-2 and C-3, the apparent fumarase rate was set to 5 (see
Ref. 10). The enrichments of PEP carbons were T1 = 1.1% and, as
determined above, T2 = 1.1% and T3 = 35 ± 0.6%;
the enrichments of oxalacetate carbons C-2 and C-3 were 25.3 ±
0.4%, determined from the values of glutamate C-3 and C-2
().
Fluxes of Glucose Metabolism
The flux
parameters, in hexose equivalents, are as follows. For cytosol: 1)
V(G) (flux of glucose inflow into the maize root tips,
normalized to 1 for calculation of relative fluxes, i.e.V(G) = 1); 2) Suc (flux of synthesis and
degradation of sucrose via sucrose phosphate synthase and invertase);
3) SS (accumulation of soluble carbohydrates in the maize root
tip); 4) Pent (synthesis of pentans with CO production from glucose C-6); 5) Pol (hexose phosphate
output for polysaccharide synthesis); 6) TFc (resynthesis of
hexose phosphates from triose phosphates); 7) FT (glycolytic
degradation of hexose phosphates to triose phosphates in the cytosol);
8) Tlc (exchange of the C-4 to C-6 moiety of cytosolic
fructose phosphate and free triose phosphates through the transaldolase
reaction); 9) Pl (hexose phosphate transport from cytosol to
plastid).
C]glucose (C-1 =
11.1%). Flux A had to be included because no fit of the
experimental data was found when the model assumed that three pentose
phosphates were either recycled to two fructose 6-phosphates and 1
triose-phosphate (as in most models) or all degraded to triose
phosphates. In the first case, the flux of triose phosphates to PEP and
the tricarboxylic acid cycle was estimated to be 0; this hypothesis was
rejected because it does not account for the labeling of the amino
acids aspartate and glutamate. In the second case, the simulation of a
[2-
C]glucose labeling resulted in an enrichment
of the plastidial hexose phosphate C-1 of only 4.5%, whereas the
experimental value is equivalent to 11.1% (). Flux A derives from a particular pool of fructose phosphate originating
from the pentose phosphate pathway, which is not in equilibrium with
the glycolytic fructose 6-phosphate. The flux of hexose phosphates
formed in the pentose phosphate pathway and immediately degraded to
triose phosphates is 2P/3-A. 13) Tlp (exchange of the
C-4 to C-6 moiety of plastidial fructose phosphate with free triose
phosphate through the transaldolase reaction); 14) Glyco (total glycolytic flux of triose phosphates to both tricarboxylic
acid cycle and fatty acid synthesis); 15) TCA (flux of PEP
into the tricarboxylic acid cycle by both the PEP carboxylase and the
pyruvate kinase reactions); 16) Lip (flux of PEP to fatty acid
biosynthesis).
Equations of Hexose Metabolism
Isotopic
steady state equations for free glucose are as follows.
CO
evolution from
[1-
C]glucose and
[6-
C]glucose are equal; then, from Equation 25,
C]glucose, with a (`) added for carbons
labeled from [6-
C]glucose; V
and V
are the fluxes of citrate synthase
and PEP carboxylase normalized to TCA = 1 as in Ref.
10. TCA is the glycolytic carbon input into the tricarboxylic
acid cycle. Equation 26 was formulated as carbon enrichment rather than
C-specific activities. After labeling with
[1-
C]glucose, enrichment values of cytosolic
hexose phosphate C-6 (H6) and plastidial hexosyl unit C-1 (G1) were
taken from , and that of triose phosphate C-3 (T3) was
calculated from P3 and V
as described above. After
labeling with [6-
C]glucose, the enrichments of
supplied glucose C-1 and C-6 were S`1 = 0, S`6 = 0.98;
that of triose phosphate C-3 was T`3 = 0.49, which assumes
negligible dilution by the pentose phosphate pathway. The enrichments
of cytosolic hexose phosphates were calculated from Equations 1, 6, 7,
and 12, which gave H`6 = 0.76 and H`1 = 0.12; since the
ratios G1/H1 and G`1/H`1 must be equal, then G`1 = 0.1. The
values of O4 and O`4 depend on the enrichment of P3 and of the CO
fixed by PEP carboxylase; the enrichment of CO
was
not determined but was found to be identical with both
[1-
C] and [6-
C]glucose.
Given this, the model of Ref. 10 shows that, for CO
enrichments varying between 0.05 and 0.2, the difference O4-O`4
remains in the range -0.036 ± 0.002; this mean value was
used.
Table:
Steady state C enrichments of
carbohydrate and amino acid carbons after incubation of maize root tips
with [1-
C]glucose
H and
C spectra as described
under ``Experimental Procedures.'' Results are given as mean
± S.D. (n = 3).
Table:
Steady state-specific radioactivities of
soluble carbohydrates (glucose, sucrose, and starch) and of C-1 of
glucose and starch after incubation of maize root tips with
[2-C]glucose
C]glucose (16 ± 0.2
dpm
nmol
); the specific radioactivities of
glucose, sucrose, and of starch glucosyl were determined as described
under ``Experimental Procedures.'' Specific radioactivities
of C-1 of free glucose and of starch glucosyl were determined by
enzymatic decarboxylation and counting the radioactivity incorporated
in CO
. Equivalent enrichment of C-1 was calculated from the
equation: E(%) = specific radioactivity of C-1 * 98/16
+ 1.1 where 98 is the enrichment (%) of labeled glucose above
natural abundance in an experiment where maize root tips would be
incubated with [2-
C]glucose (99% isotopic
enrichment), and 16 (dpm
nmol
) is the actual
specific radioactivity of the labeled substrate. Results are the mean
(±S.D.) of three determinations from two independent
experiments. ND, not determined.
Table:
Relative and absolute values of metabolic
fluxes in maize root tips incubated with 200 mM glucose
h
tip
). Absolute
fluxes are given in nmol hexose
equivalent
h
tip
. Some
fluxes were determined directly. The rate of glucose uptake V(G) was 215
nmol
h
tip
, and was
normalized to 1, i.e. V(G) = 1. The rate soluble sugar
accumulation, SS, was determined from the change in glucose
and fructose levels, which were increasing linearly around 12 h at a
rate of 39 nmol
h
tip
,
whereas sucrose had reached a plateau after 5 h (Fig. 7). The mean rate
of starch accumulation over 12 h, St, was only 3 nmol of
hexose
equivalent
h
tip
. The
accumulation of fatty acids, Lip, was estimated from the
increase in dry weight (0.038
mg
h
tip
), assuming
that the lipid content per mass unit (18) remained constant; Lip was 7 nmol of hexose
equivalent
h
tip
. The
rate of CO
evolution, V(CO
),
was 230 nmol
h
tip
(18). Flux values calculated from the model based upon carbon
enrichment studies are shown as the mean and range of separate
simulations using extreme values of enrichments (see Suc determination in Fig. 8).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.