In vivo respiratory metabolism of illuminated leaves.

Abstract

Day respiration of illuminated C(3) leaves is not well understood and particularly, the metabolic origin of the day respiratory CO(2) production is poorly known. This issue was addressed in leaves of French bean (Phaseolus vulgaris) using (12)C/(13)C stable isotope techniques on illuminated leaves fed with (13)C-enriched glucose or pyruvate. The (13)CO(2) production in light was measured using the deviation of the photosynthetic carbon isotope discrimination induced by the decarboxylation of the (13)C-enriched compounds. Using different positional (13)C-enrichments, it is shown that the Krebs cycle is reduced by 95% in the light and that the pyruvate dehydrogenase reaction is much less reduced, by 27% or less. Glucose molecules are scarcely metabolized to liberate CO(2) in the light, simply suggesting that they can rarely enter glycolysis. Nuclear magnetic resonance analysis confirmed this view; when leaves are fed with (13)C-glucose, leaf sucrose and glucose represent nearly 90% of the leaf (13)C content, demonstrating that glucose is mainly directed to sucrose synthesis. Taken together, these data indicate that several metabolic down-regulations (glycolysis, Krebs cycle) accompany the light/dark transition and emphasize the decrease of the Krebs cycle decarboxylations as a metabolic basis of the light-dependent inhibition of mitochondrial respiration.

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In Vivo Respiratory Metabolism of Illuminated Leaves1
Guillaume Tcherkez*, Gabriel Cornic, Richard Bligny, Elizabeth Gout, and Jaleh Ghashghaie
Laboratoire d’Ecophysiologie Ve ´ge ´tale, Unite ´ Mixte de Recherche 8079, Ba ˆt. 362, Centre scientifique d’Orsay,
Universite ´ Paris XI, 91405 Orsay cedex, France (G.T., G.C., J.G.), and Laboratoire de Physiologie Cellulaire
Ve ´ge ´tale, Unite ´ Mixte de Recherche 5168, Commissariat a ` l’Energie Atomique-Grenoble,
38054 Grenoble cedex 09, France (R.B., E.G.)
Day respiration of illuminated C3leaves is not well understood and particularly, the metabolic origin of the day respira-
tory CO2production is poorly known. This issue was addressed in leaves of French bean (Phaseolus vulgaris) using12C/13C
stable isotope techniques on illuminated leaves fed with13C-enriched glucose or pyruvate. The13CO2production in light was
measured using the deviation of the photosynthetic carbon isotope discrimination induced by the decarboxylation of the
13C-enriched compounds. Using different positional13C-enrichments, it is shown that the Krebs cycle is reduced by 95% in the
light and that the pyruvate dehydrogenase reaction is much less reduced, by 27% or less. Glucose molecules are scarcely
metabolized to liberate CO2in the light, simply suggesting that they can rarely enter glycolysis. Nuclear magnetic resonance
analysis confirmed this view; when leaves are fed with13C-glucose, leaf sucrose and glucose represent nearly 90% of the leaf
13C content, demonstrating that glucose is mainly directed to sucrose synthesis. Taken together, these data indicate that several
metabolic down-regulations (glycolysis, Krebs cycle) accompany the light/dark transition and emphasize the decrease of the
Krebs cycle decarboxylations as a metabolic basis of the light-dependent inhibition of mitochondrial respiration.
Illuminated leaves simultaneously assimilate CO2
through the photosynthetic carbon reduction cycle
and lose CO2through photorespiration and day res-
piration. In darkness, leaves no longer assimilate CO2
via the photosynthetic carbon reduction cycle but pro-
duce CO2through dark respiration. Although dark
respiration is known to involve glycolysis and CO2
production through pyruvate dehydrogenation and
the degradative Krebs cycle (Trethewey and ap Rees,
1994; Plaxton, 1996), the carbon metabolism that is
responsible for the CO2respiratory release in the light
is almost unknown. This is so because the day re-
spiratory CO2flux is very low and masked by the
photosynthetic carbon fixation and the photorespira-
tory CO2production in the light, and is thus difficult to
study.
Nevertheless, it has been repeatedly shown, using
either the Laisk’s (Laisk, 1977) or Kok’s method (Kok,
1948), that the rate of day respiration (Rd) is less than
that of dark respiration (Rn; for review, see Atkin et al.,
2000) so that light is known to inhibit respiration, with
a Rd/Rnvalue (usually denoted as m) ranging from
30% to 100% (for a recent study, see Peisker and Apel,
2001). Pioneering gas exchange measurements on
mustard suggested that some enzymatic activities are
inhibited in the light so that substrates accumulate
(Cornic, 1973), explaining the respiratory burst when
leaves are darkened: the light enhanced dark respira-
tion. More recently, it has been shown in the unicellu-
lar alga Selenastrum minutum that pyruvate kinase (Lin
et al., 1989) is inhibited by light. It is also the case of the
pyruvate dehydrogenase complex that is partly in-
activated by (reversible) phosphorylation in extracts
from illuminated leaves (Budde and Randall, 1990;
Tovar-Mendez et al., 2003). Photorespiration is also
probably involved in the inhibition of pyruvate de-
hydrogenase as it has been shown that this enzyme is
down-regulated by NH3, which is a byproduct of the
photorespiratory Gly decarboxylation (Kro ¨mer, 1995).
Enzymes of the Krebs cycle are also assumed to be
inhibited in the light because of a high mitochondrial
NADH level due to photorespiratory Gly decarboxyl-
ation (Atkin et al., 2000). Additionally, it has been
shown that the mitochondrial isocitrate dehydroge-
nase is inhibited by the high NADPH/NADP ratios
that occur in the light (Igamberdiev and Gardestro ¨m,
2003).
Although all these enzymatic data suggest that the
respiratory pathway is down-regulated in the light
regarding both glycolysis and the Krebs cycle, respi-
ratory metabolic fluxes in vivo in leaves are not well
known. Some labeling experiments with carbon iso-
topes (13C or14C) have already been done to disentan-
gle respiratory metabolic fluxes in vivo in the light and
in the dark, but surprisingly, studies that have focused
on labeling of the resulting respired CO2are scarce.
Using14CO2labeling techniques, Pa ¨rnik et al. (2002)
suggested that CO2production in the light is com-
posed of (1) decarboxylation of primary products like
triose phosphates and malate (between 10% and 50%
1This work was supported by the European Community’s
Human Potential Programme (grant no. HPRN–CT–1999–00059,
NEtwork for Terrestrial ecosystems CARbon Budget, to J.G.) and by
the Centre National de la Recherche Scientifique (to G.C. and R.B.).
* Corresponding author; e-mail guillaume.tcherkez@ese.u-psud.
fr; fax 33–169153424.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.105.062141.
This article is published in Plant Physiology Online, Plant Physiology Preview Section, which publishes manuscripts accepted for
publication after they have been edited and the authors have corrected proofs, but before the final, complete issue is published
online. Early posting of articles reduces normal time to publication by several weeks.
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from one species to another), and (2) decarboxylation
of end-products like Suc and starch to a greater extent
(50%–90%).
atmosphere has been used to show that day respira-
tion is less than night respiration, and its rate as well as
the ratio m decreased at high CO2concentration (Pinelli
and Loreto, 2003). It has thus been proposed that
inhibition of CO2production is determined by the CO2
fixation flux (Atkin et al., 1998; Pinelli and Loreto,
2003). The ability of leaves to oxidize some metabolites
through respiration has also been investigated with
feeding experiments using labeled organic com-
pounds. Supplying13C-enriched Glc to myrtle leaves
in the light with CO2-free air did enrich the CO2
subsequently respired in the dark, although the CO2
was not completely labeled (Affek and Yakir, 2003).
Nevertheless, the13C amount in the light-respired CO2
was not measured in that study.
So already published data do not show what the
respiratory metabolic pathway is actually in illumi-
nated leaves; that is, what the metabolic fluxes asso-
ciated with the day respiratory CO2production are.
We address this question here by feeding illuminated
French bean (Phaseolus vulgaris) leaves with positional
13C-enriched Glc and pyruvate and measuring the
resulting
respired CO2and in intermediary respiratory com-
pounds, using isotope ratio mass spectrometry and
nuclear magnetic resonance (NMR), respectively. The
isotopic analysis of CO2respired in the dark and the
measurement of the carbon isotope discrimination
during photosynthesis allows us to calculate the
13C-content in dark- and light-respired CO2, respec-
tively. Respiration of supplied13C1- and13C3-enriched
Glc is found to be almost completely inhibited by light.
By contrast, decarboxylation of13C1-enriched pyruvate
through the pyruvate decarboxylation is much less
inhibited by light, unlike13C-2-pyruvate. These data
are supported by NMR spectra obtained from illumi-
nated leaves and taken as a whole, they suggest that
(1) the Krebs cycle and glycolysis are strongly inhib-
ited by light, with little interconversion between triose
phosphates and hexose phosphates through the triose
phosphate isomerase and aldolase reactions, and (2)
the pyruvate dehydrogenation is only partly inhibited
by light, with the acetyl-CoA molecules being directed
toward purposes other than respiration.
12CO2(respiratory) production in a
13C
13C-enrichment in both day and dark-
THEORY
This ‘‘Theory’’ section describes the mathematical
background used to calculate the Rddecarboxyla-
tions when detached leaves were supplied with
13C-enriched molecules (Fig. 1).
In the following, the isotope composition (d13C) and
the isotope ratio (13C/12C) are denoted as d and R,
respectively, and the percentage of13C is denoted as l.
l is simply deduced from d through the following
relationship:
l5
13C
13C112C5
13C=12C
13C=12C115
R
R11:
As d5R2Rst
Dee reference material (Rst5 0.0112372), we have:
Rst
where Rstis the13C/12C ratio in the Pee
l5
1
11
1
Rstðd11Þ
:
ð1Þ
Rate of13C-Enriched Substrate Decarboxylation
in the Light (rday)
The on-line discrimination value was obtained us-
ing the method of Evans et al. (1986):
Dobs5
jðde2doÞ
11do2jðde2doÞ;
where the isotope compositions of air entering and
leaving the cuvette are deand do, respectively. j is
equal to ce/(ce2 co) where ceand coare the CO2molar
fractions in the entering and leaving air, respectively.
The Dobsvalue obtained before the substrate addi-
tion is denoted as D. It is assumed that after substrate
addition, the air leaving the leaf cuvette is the sum
of the CO2left by photosynthetic discrimination
and additional CO2released from substrate respira-
tion. The latter flux is denoted as rday. If the leaf area
is denoted as S and cfixedis the CO2amount (in mL/L)
Figure 1. Metabolic model used in this paper for decarboxylations of
13C-enriched substrates and respiratory variables taken into account
(rPDHand rKand the sum, rdayor rnight). rPDHand rKare the rates of
decarboxylation of13C-enriched substrates through the PDH reaction
and the Krebs cycle, respectively. rdayand rnightare the sum of rPDHand
rKin the light and in the dark, respectively. Different symbols are used
for different carbon atom positions in order to see the pathways to CO2
production.
Tcherkez et al.
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fixed by photosynthesis, the mass balance equation for
CO2is as follows:
ce1rday
d
where d (L/s) is the air flow through the leaf cuvette
and VMthe molar volume. Thus, we have:
cfixed5ce1rday
SVM5cfixed1co;
d
SVM2co:
ð2Þ
Case of Feeding with a Compound That Has
a Homogeneous Isotope Composition
Homogeneous isotopic compounds arenot involved
in this study, but the calculation is explained here as it
gives a basis to understand the next step, that is, the
use of compounds with a heterogeneous isotope com-
position.
The isotope composition of the substrate fed to the
leaf is denoted as ds. Isotopic mass balance is so that
the
from the additional CO2release (rdaySVMls/d) is equal
to the sum of the13C amount fixed by the leaf (cfixed
lfixed) and the13C amount leaving the cuvette (colo).
That is,
13C amount entering the cuvette (cele) plus that
cele1rday
d
SVMls5cfixedlfixed1colo:
ð3Þ
Note that the similar relationship with deltas is not
correct as the strong
one to neglect R compared to 1 so that l 6¼ R. lfixedis
obtained with the usual relationship dfixed5do2D
Equation 1, where D is obtained before feeding by on-
line isotopic measurements (Evans et al., 1986).
Substituting Equation 2 in Equation 3 gives:
13C-enrichment does not allow
D11and
rday5
d
SVM
3colo2cele1ðce2coÞlfixed
ls2lfixed
:
ð4Þ
It should be noted that possible12C/13C fractiona-
tions that can occur during the absorption of labeled
compounds are neglected here. The effect of such
a fractionation is clearly negligible, that is, in the per
mil order of magnitude, compared to the labeling
level, which is in the percent order of magnitude or
more.
Case of Feeding with a Compound That Has a
Nonhomogeneous Positional Isotope Composition
The underlying assumption of the previous para-
graph is that the substrate is isotopically homoge-
neous. However, the isotope composition of the
feeding substrates used in these experiments is non-
homogeneous so that its different carbon atom posi-
tions do not have the same D values. In such a case, the
metabolic reactions that are responsible for the de-
carboxylation of the carbon atoms and their rates
should be taken into account. For example, this occurs
when Glc or pyruvate is added: the C-1 atom of
pyruvate is decarboxylated by pyruvate dehydroge-
nase, while the C-2 and C-3 positions are decarboxy-
lated by the Krebs cycle (Fig. 1). Similarly, the C-3 and
C-4 atom positions of Glc are decarboxylated by the
pyruvate dehydrogenase reaction, the other being
decarboxylated by the Krebs cycle. Advantage can
then be taken from this with positionally13C-enriched
substrates;
the CO2produced by pyruvate dehydrogenase, while
13C-2-pyruvate would specifically enrich the CO2that
comes from the Krebs cycle. The same applies to
positional13C-enrichment in Glc.
The additional decarboxylations through the pyru-
vate dehydrogenase (PDH) reaction and the Krebs
cycle are denoted as rPDHand rK, respectively (Fig. 1).
With the relationship rday5 rPDH1 rK, Equation 2 still
works. For13C-1-pyruvate, Equation 3 becomes:
13C-1-pyruvate would specifically enrich
cele1SVM
d
ðl1rPDH1lcrKÞ5cfixedlfixed1colo;
where l1is the13C percentage in the C-1 position of
the labeled pyruvate. lcis the13C percentage of the
other (unlabeled) positions. For
have:
?
where l2is the
position of pyruvate. lc(unlabeled positions) is the
same as lcof Equation 5 (13C-1 enrichment). It can be
seen that the two conditions of
two Equations (5 and 6) so that rPDHand rKcan be
deduced by a substitution procedure. A similar pro-
cedure is used for the positional13C-enrichment of Glc.
ð5Þ
13C-2-pyruvate, we
cele1SVM
d
lcrPDH1lc1l2
2
rK
?
5cfixedlfixed1colo; ð6Þ
13C percentage in the C-2 (labeled)
13C-enrichment give
Rate of13C-Enriched Substrate Decarboxylation
in Darkness
The CO2that is produced in darkness after a light
period with
from respiratory oxidation of new photosynthates
(13C percentage in the fixed carbon lfixed), photosyn-
thates from the previous light period in the green-
house (13C percentage lprevious), and additional C
coming from the13C-enriched substrate fed to the leaf
(13C percentage ls). It has been previously shown that
the contribution of new photosynthates to dark respi-
ration after 3-h light in French bean is 40% (Nogue ´s
et al., 2004). So the13C percentage in photosynthates
feeding respiration is given by lp5 0.4 lfixed1 0.6
lprevious. It should be noted that possible variations
in the coefficients due to some physiological reasons
do only introduce a minor error in the estimate of the
13C-enriched substrate decarboxylation rnightbecause
of the strong13C-enrichment in the substrate.
The total CO2production in the dark is denoted as
Rn. The13C percentage in dark respired CO2(denoted
as lglobal) is calculated with the d13C value and Equa-
tion 1. The
relationship:
13C-enriched substrate feeding comes
13C mass balance gives the following
Day Respiratory Metabolism of Leaves
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Rnlglobal5rnightls1ðRn2rnightÞlp:
Rearranging, it gives:
ð7Þ
rnight5Rn3lglobal2lp
ls2lp
:
ð8Þ
When substrates do not have a homogeneous iso-
topic distribution (positional enrichment), Equation 7
is completed to:
Rnlglobal5ðrPDHl11rKlcÞ1ðRn2rPDH2rKÞlp
for the13C-1-pyruvate feeding. And similarly, for the
13C-2-enrichment, it gives:
?
ð9Þ
Rnlglobal5 rPDHlc1rK3lc1l2
2
?
1ðRn2rPDH2rKÞlp:
ð10Þ
Equations 9 and 10 allow one to extract rPDHand rK
with a substitution procedure. The method is similar
for13C-Glc.
RESULTS
On-Line Carbon Isotope Discrimination of Leaves Fed
with13C-Enriched Substrates
The carbon isotope discrimination during photo-
synthesis of detached French bean leaves before and
after addition of13C-enriched carbohydrates is shown
in Figure 2. Before adding substrates, the carbon
isotope discrimination was around 20& in all cases.
This is in accordance with the pi/pavalue around
0.7 (data not shown). At t 5 60 min,
substrates were added. The overall signature of Glc
was 5,500& and that of pyruvate was 2,500&. When
13C1-pyruvate was supplied, the carbon isotope dis-
crimination then increased to Dobs5 180& as a con-
sequence of
The carbon isotope discrimination then slightly de-
creased and reached approximately 140&. The on-line
carbon isotope discrimination value increased much
less with
value of Dobsaround 50&, indicating that decarbox-
ylations following Pyr dehydrogenation (i.e. Krebs
cycle) had very small rates. Glc was hardly decar-
boxylated in the light, thus with a very small increase
in Dobs(up to 35&–40& only).
13C-enriched
13C1-enriched pyruvate decarboxylation.
13C2-enriched pyruvate, with a maximum
Rates of Decarboxylation in the Light
The rates of decarboxylation of the
substrates (see Fig. 1 that summarizes the parameters
considered) in the light were calculated using the
carbon isotope discrimination Dobsand are given in
Table I. These calculations used a two variable model
(see ‘‘Theory’’ section) that is, rPDH, the rate of de-
carboxylation through the PDH, and rK, the decarbox-
ylation through the Krebs cycle. These rates may be
13C-enriched
compared to the overall Rd. As expected from con-
clusions of Figure 2, the decarboxylation rate of
pyruvate through the PDH reaction was around 0.05
to 0.06 mmol m22s21, the other decarboxylation rates
being very low, under 0.01 mmol m22s21. Although the
effect of decarboxylation of
striking in Figure 2, the decarboxylation rate was low
compared to day respiration (Rd, around 0.6 mmol m22
s21) simply because the labeling was strong in Pyr. It is
noteworthy that the decarboxylation rates were neg-
ligible compared to the overall Rd, so that we argue
that the respiratory pathway was not artefactually
enhanced in our feeding experiment. Accordingly,
the overall Rdof fed leaves was similar to that of the
control. Nevertheless, the decarboxylation CO2flux
could have been somewhat underestimated because of
refixation (see below).
13C1-enriched Pyr was
Rates of Decarboxylation in Darkness
After 180 min (see Fig. 2), light was switched off and
the carbon isotope composition of the CO2evolved in
the first 30 min of darkness was measured. The d13C
value of respired CO2with
ing was approximately 740& and 640&, respectively,
and the d13C value of respired CO2with13C1- and
13C2-pyruvate feeding was approximately 520& and
290&, respectively (Fig. 2, right). The decarboxylation
rates calculated using these values are shown in Table I.
rPDHwas around 0.070 mmol m22s21with either Glc
or pyruvate and rKwith Glc was around the double
(0.2 mmol m22s21) of that with pyruvate. Clearly, these
decarboxylations witness that leaves oxidized mole-
cules via glycolysis in the dark. A similar result was
already obtained by Stitt and ap Rees (1978) with
14CO2. When compared to the day decarboxylation
values, the inhibition by light was around 95% for both
rPDHand rKwith Glc and around 30% and 95% for rPDH
and rKwith pyruvate, respectively. In other words,
light inhibited only partially the pyruvate dehydroge-
nase reaction and almost stopped the Krebs cycle. In
the light, the respiratory breakdown of Glc into CO2
did not occur through the Krebs cycle or the pyruvate
dehydrogenase reaction, simply demonstrating that
the Glc molecules could not reach these metabolic
steps in illuminated leaves.
13C1- and
13C3-Glc feed-
Metabolic Pathways That Consumed13C-Enriched Glc
The weakness of Glc or pyruvate decarboxylation in
the light raises the question of the fate of these
molecules in the leaf. That is why starch purification
on sample of the experiment of Figure 2 was made and
NMR analysis of illuminated leaves fed with posi-
tional, fully13C-labeled substrates (99%13C in a given
position) was done. The results are shown in Table II
and Figure 3, respectively.
13C-enriched Glc supplied to leaves was directed to
Suc synthesis; the
13C content of Suc as a whole
Tcherkez et al.
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approached 60% in both (C-1 and C-3) enrichment
conditions (Fig. 3, A and B). When13C3-Glc was used,
the scrambling of carbon atoms appeared, with a small
labeling of all the carbon atom positions in the Glc
moiety of Suc (13C content around 4%). This presum-
ably came from the pentose phosphates cycle. Accord-
ingly, this effect was not seen with13C1-Glc because the
first steps of the cycle are the dehydrogenation and
decarboxylation of the C-1 carbon atom of Glc.
Surprisingly, starch was also labeled by Glc (Table
II); with13C1-Glc and13C3-Glc, the d13C value of starch
was strongly higher (21.9& and 29&, respectively)
than in unfed leaves (231.4&). In other words, Glc
could feed starch synthesis. Its contribution should
have nevertheless been low as the starch amount was
not significantly different between fed and unfed
leaves (Table II). The contribution of feeding Glc (in
percent of total starch amount), denoted here as s, can
be calculated with the following mass-balance rela-
tionship:
d13Cwithfeeding5s3d13CfeedingGlc1ð12sÞ3d13Cwithoutfeeding
Figure 2. Left, Development of the on-line carbon isotope discrimination (Dobs) of detached bean leaves before and after feeding
with Glc that is13C-enriched in C-1 ()) or C-3 (h), or pyruvate that is13C-enriched in C-1 (;) or C-2 (:). In both positional
enrichment cases, the overall d13C values of Glc and pyruvate are 5,500& and 2,500&, respectively. The detached leaves are
first put in distilled water and the light is turned on. The vertical dotted line indicates the moment at which the substrate (Glc or
pyruvate) is supplied (t 5 60 min). The horizontal dotted line represents the mean photosynthetic fractionation before substrate
feeding (Dobsapproximately equal to 20&). The gas exchange conditions were 350 mL L21CO2in 21% O2, 22?C, and 450 mmol
m22s21light. Note that the Dobsmeasurements only begin after photosynthesis stabilizes that is, after approximately 30 min in
the light. At t 5 180 min, the light is switched off for dark-respired CO2measurements. The trends of the on-line isotope
discrimination with Pyr feeding are indicated with a solid line. Right, d13C value (in per mil) of the CO2respired in the dark just
after having switched off the light. Same symbols as for the left section.
Table I. Respiration rate and calculated decarboxylations (in mmol m22s21) of Glc and pyruvate supplied to detached French bean leaves
through the PDH and the Krebs cycle in the light and in the dark (see ‘‘Theory’’ section for calculation details)
Day respiration rates were measured as the slope of G/riRnrelationships (see ‘‘Gas Exchange Measurements’’ section) and are thus total day
respiratory fluxes in the light. Decarboxylation data are mean and SE of three independent measurements. The mean inhibition of decarboxylation by
light was calculated as the ratio mean light decarboxylation/mean dark decarboxylation.
ControlPyruvateGlc
Light
Rd
rPDH
rK
Darkness
Rn
rPDH
rK
Day respiration rate
Decarboxylation from the PDH reaction
Decarboxylation from the Krebs cycle
0.632 6 0.125 0.625 6 0.150
0.058 6 0.009
0.005 6 0.004
0.539 6 0.143
0.004 6 0.001
0.009 6 0.004
Dark respiration rate
Decarboxylation from the PDH reaction
Decarboxylation from the Krebs cycle
1.178 6 0.0391.266 6 0.071
0.079 6 0.023
0.116 6 0.011
1.331 6 0.115
0.070 6 0.015
0.236 6 0.050
iPDH
iK
Mean inhibition of the PDH reaction by light
Mean inhibition of the Krebs cycle by light
27%
95%
94%
96%
Day Respiratory Metabolism of Leaves
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that gives s 5 0.5% 6 0.2% and 0.4% 6 0.2% for
13C1-Glc and13C3-Glc, respectively. This contribution
was very low, but was clearly seen in starch (Table
II) simply because the d13C value of the labeling Glc
was very high. This result indicates that Glc mole-
cules from the cytoplasm (or, more generally, C from
the fed Glc) could reach the chloroplastic compart-
ment and enter the starch synthetic pathway.
The enriched carbon atoms of Glc were almost not
redistributed to other positions by metabolic path-
ways; when supplied with
atoms in leaf Glc, Fru, or Suc were not labeled (Fig.
3A). Similarly, when supplied with13C3-Glc, the C-4 car-
bon atoms in leaf Glc and Fru were not labeled and the
corresponding positions in Suc were only weakly
labeled (4% of the13C content; Fig. 3B). In other words,
the scrambling of carbon atoms through the triose
phosphates and hexose phosphates interconversion
(with triose phosphate isomerase and aldolase) was
very low. This result is consistent with the gas ex-
change measurements of Figure 2 in which glycolysis
appeared to be stopped so that Glc was not (or very
weakly) decarboxylated.
13C1-Glc, the C-6 carbon
Metabolic Pathways That Consumed
13C-Enriched Pyruvate
When leaves were fed with13C2-Pyr (99% of13C in
C-2), Ala was (weakly) labeled in C-2, indicating that
Pyr had been aminated and citrate was labeled,
strongly suggesting that some of the Pyr molecules
entered the Krebs cycle (Fig. 3D). Surprisingly, when
fed with13C1-Pyr, leaves had only a few labeled carbon
atoms (Fig. 3C). Ala appeared as only weakly labeled
in C-1, but this originated mainly from the low de-
tectability of carboxyl (2COOH) carbon atoms with
NMR. Moreover, the CO2produced in the light ac-
counted for an important
boxylation rate of 0.05 mmol m22s21(Table I), there
was a13C loss of 350 mmol13C m22during the light
treatment (2 h),while approximately 1,950 mmol Pyr
m22entered the leaf (5Pyr concentration 3 transpi-
ration rate 5 15 mol L213 18 mL m22s213 2 h light),
13C loss; using a decar-
that is, approximately 18% of the13C supplied in C-1.
A similar calculation gives, with13C2-Pyr, a13C loss of
only approximately 1%.
Refixation of Decarboxylated CO2
The rate of the decarboxylation of
substrates in the light may have been underestimated
by possible refixation of CO2. If so, the absolute rate of
refixation is expected to be important when13C1-Pyr
was supplied to leaves as decarboxylation was high in
that case (Table I). Nevertheless, Glc and Fru were not
labeled and Suc was only weakly labeled in the C-3
position of the Fru moiety (Fig. 3C). Although it is
difficult to quantify such small quantities, this labeling
in Suc accounted, as a maximum, for 0.5 mmol g21of
13C, that is, a13C (re)fixation rate of 0.009 mmol m22s21
during 2 h of the treatment in the light. When13C1-Pyr
was supplied to leaves, starch was also labeled (the
isotope signature is around 220&, compared to 231&
expected; Table II) and this was also a consequence
of refixation. The amount of
in starch was calculated to be 0.2% (similar calcula-
tion as for section ‘‘Metabolic Pathways That Con-
sumed13C-Enriched Glc’’). The starch amount and the
percentage of carbon in starch were 0.015 mg mg21
fresh weight and approximately 40%, respectively.
This gives a refixation rate (of labeled carbon) of
0.008 mmol m22s21. The overall refixation rate would
thus have been, as a maximum, 0.017 mmol m22s21.
This means that the total decarboxylation rate of
13C1-Pyr would have been 0.058 (see Table I) 1 0.017 5
0.075 mmol m22s21and so, the inhibition of the
PDH reaction by light would have been approxi-
mately 5% only. The effect of refixation in the other
case (13C2-Pyr) was clearly negligible: starch was not
labeled (Table II) and no labeling could be seen in Suc,
Glc, or Fru (Fig. 3D). As the rates of decarboxylation
were similar with C-1 and C-3
refixation was likely to be negligible in both cases.
13C-enriched
13C-enriched carbon
13C-enriched Glc,
DISCUSSION
The respiratory metabolism in illuminated leaves
isinhibitedcomparedtodarkness,asrepeatedlyshown
by gas-exchange measurements (for review, see Atkin
et al., 2000). However, the metabolic basis of such an
inhibition is not well known. We addressed this ques-
tion by feeding experiments using13C-enriched sub-
strates and followed the13C atoms with isotope ratio
mass spectrometry and NMR to determine which
metabolic pathways are inhibited in the light.
The Pyruvate Dehydrogenase Activity in the Light
The two main steps responsible for the respiratory
CO2production are the dehydrogenation of Pyr (PDH
reaction) and the Krebs cycle (Fig. 1). The carbon
atoms that are decarboxylated are not the same in
Table II. Amount and carbon isotope composition (d13C) of starch
from detached leaves supplied with13C-enriched Glc (d13C 5,500&)
or13C-enriched Pyr (d13C 2,500&), 15 mmol L21for 2 h in the light
with 350 mL L21CO2at 251& (see Fig. 1) after 1 h in the light
without feeding
Data are mean and SD of three independent measurements.
Conditions Amount
d13C
mg mg21fresh weight
13.1 6 5.3
15.1 6 5.0
15.0 6 3.3
13.6 6 2.9
11.7 6 5.4
12.2 6 3.2
&
Control (greenhouse)
No feeding
13C1-Glc
13C3-Glc
13C1-Pyr
13C2-Pyr
228.5 6 0.5
231.4 6 1.3
21.9 6 9.0
29.0 6 9.5
216.5 6 3.4
229.9 6 1.6
Tcherkez et al.
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both: the PDH reaction decarboxylates the C-1 of Pyr,
while the Krebs cycle decarboxylates the two others.
Feeding illuminated leaves with
in C-1 significantly enriched the respiratory CO2
produced in the light, as revealed by the strong
modification of the photosynthetic carbon isotope dis-
crimination measured on-line, from the steady value
of 20& to approximately 160& (Fig. 2). Clearly, the
PDH reaction consumes the Pyr molecules, and so is
not totally inhibited in the light. When compared to
the dark decarboxylation rate, the calculatedinhibition
is 27% (Table I). Refixation of decarboxylated CO2can
nevertheless occur and lead to an overestimation of the
13C-enriched Pyr
inhibition level. Indeed, there was a small13C enrich-
ment in Suc (Fig. 3) as well as in starch (Table II) and
a calculation (see ‘‘Results’’) gives a refixation rate of
0.017 mmol m22s21; that is, a total decarboxylation rate
of Pyr of 0.075 mmol m22s21. The refixation rate
obtained here stands for 22% of the (total) decarbox-
ylation of
dance with calculations that use14C data by Gerbaud
and Andre ´ (1987) on sunflower leaves (between 15%
and 22%), but a little low compared to Pinelli and
Loreto (2003) who found a refixation rate of 40% in
mint leaves maintained at 350 mL L21. In this study,
we looked at only
13C-enriched Pyr, a value that is in accor-
13C enrichment in the two main
Figure 3. Distribution of13C from NMR anal-
ysis of leaves fed with13C1- or
and B),13C1- or13C2-pyruvate (C and D) for
2 h in the light at ambient CO2. In each case,
the compound supplied to leaves is indicated
in the top left of each section. Simplified
metabolic pathways are represented in order
to emphasize the relationships between me-
tabolites. Cit, Citrate; TP, triose phosphates;
Org Ac, organic acids. The dashed line stands
for the production of organic acids from
pyruvate via the phosphoenolpyruvate car-
boxylation. The bottom continuous line in C
and D stands for the refixation of decarbox-
ylated CO2. Carbon atoms are colored in
accordance with their labeling level: black,
no labeling, gray, low labeling, white, strong
labeling. For each labeled carbon, numbers
are in percent of
Carbon atoms that are considered to be
labeled are those which proportion of
(13C/12C 113C) is equal or more than 2% (the
natural abundance of
1.1%).
13C3-Glc (A
13C that is in the sample.
13C
13C is approximately
Day Respiratory Metabolism of Leaves
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compounds of CO2fixation; that is, Suc and starch.
Nevertheless, we recognize that some
other compounds were missed, leading to a small
underestimation of the refixation rate. Taking our
value as correct, this would give a light inhibition
value of the PDH reaction of only 5%.
However, it is not possible in our experiment to dis-
tinguish the chloroplastic PDH activity from the mito-
chondrial one so that our values do represent the total
cellular PDH activity. It has been shown that the
mitochondrial PDH is partly inhibited in illuminated
leaves by phosphorylation (Budde and Randall, 1990).
In addition, photorespiratory produced NH3is also
assumed to inhibit this enzyme (Kro ¨mer, 1995). By
contrast, the chloroplastic PDH is not regulated by
phosphorylation and is assumed to be active in the
light (Plaxton, 1996). The decarboxylation rate mea-
sured in this study (Table I) is likely to be the sum of
both PDH activity in which the chloroplastic enzyme
plays the major role.
Furthermore, the PDH reaction may be artefactually
activated by the amount of Pyr (thermodynamic mass
action law) introduced in the leaf. We nevertheless
think that this effect is minor; the pyruvate concentra-
tion in the feeding solution is only 15 mmol L21and
although it is difficult to detect carboxyl carbon atoms
by NMR, it was not possible to see the corresponding
C-1 atom of Pyr on the NMR spectrum, indicating that
the Pyr concentration in the leaf is certainly less than
2 mmol L21in the cell.
13C atoms in
Glycolysis and the Krebs Cycle Are Inhibited
in the Light
In contrast with13C1-Pyr, when leaves were fed with
13C2-Pyr, the photosynthetic carbon isotope discrimi-
nation was hardly modified (Fig. 2), indicating that
there was nearly no decarboxylation of the C-2 carbon
atom of Pyr. In other words, the Krebs cycle activity
was very low and so there is a very small
production. The calculated decarboxylation rate is
0.005 mmol m22s21only in the light; that is, the Krebs
cycle is inhibited by 95% compared to the dark de-
carboxylation rate (Table I). This is consistent with the
NMR results: (1) the intermediary products of the
Krebs cycle such as succinate and fumarate are not
detected, Glu (that is the amino acid that corresponds
to a-ketoglutarate) was hardly labeled, (2) the two
carboxyl atoms of citrate were
citrate stood for 15% of the sample
each carboxyl carbon atom represented 7.5% only of
the
previous observations of Hanning and Heldt (1993)
that mitochondria extracted from illuminated leaves
had a low metabolic flux throughout the Krebs cycle.
In addition, the mitochondrial matrix in the light is
supposed to be reduced because of the photores-
piratory Gly decarboxylation that lead to a high
NAD(P)H/NAD(P)1ratio. Some enzymes of the
Krebs cycle are inhibited by the high NADH/NAD1
13CO2
13C-labeled so that
13C content, i.e.
13C content. These data are in accordance with
ratios (for review, see Siedow and Day, 2000), and it
has been recently found that isocitrate dehydrogenase
from pea is inhibited by high NADPH/NADP1ratios,
a feature that occurs in illuminated mitochondria
(Igamberdiev and Gardestro ¨m, 2003).
The fact that the Krebs cycle is slowed down in the
light raises the question of the fate of acetyl-CoA
molecules producedby thePDHreaction. Inour study,
acetyl-CoA probably accumulated a little: the decrease
in the photosynthetic on-line discrimination after
115 min (Fig. 2) suggests that the PDH reaction was
less active. This was likely because of the retroinhibi-
tion of Pyr dehydrogenase by its product acetyl-CoA
(Harding et al., 1970; Rapp et al., 1987), as the Krebs
cycle activity that consumes acetyl-CoA was severely
diminished in the light. A significant part of the acetyl-
CoAmoleculeswasdirectedtofattyacidproductionin
the chloroplast (Ohlroggeand Jaworski,1997). Accord-
ingly, the mutant line of Arabidopsis (Arabidopsis
thaliana) that produces the antisense RNA of the PDH
kinase (thus enhancing the mitochondrial PDH reac-
tion),accumulated14C-labeledfattyacidswhen14C-Pyr
was fed to(photosynthetic) stems (Marilliaet al.,2003),
strongly suggesting that fatty acid synthesis can act as
outfall for acetyl-CoA molecules.
13C-enriched Glc was only weakly decarboxylated in
the light whatever the position of the13C-enrichment
was (Fig. 2). Thus, Glc molecules could hardly reach
the PDH step, very likely because they could not enter
glycolysis. Instead, the Glc molecules were directed to
Suc synthesis (Fig. 3). Further, when fed with13C1-Glc,
the Fru moiety in Suc was only very weakly labeled in
C-6, and similarly, when fed with
moiety in Suc was hardly labeled in C-4 (Fig. 3).
Clearly, this shows that the C-1/C-6 and C-3/C-4
interconversion through the triose phosphates isom-
erase reaction was minor. In other words, only a small
fraction (less than approximately 5% of the13C-Glc fed
to the leaf) of the Glc molecules reached this step,
strongly suggesting a high metabolic resistance to the
glycolytic breakdown of hexoses. Noteworthy, these
results are in accordance with the regulation of
the enzymes responsible for the phosphorylation/
dephosphorylation of Fru-6-P to Fru-2,6-bisphosphate:
(1) in the light, the high triose phosphates/inorganic
phosphate decreases the Fru-2,6-bisphosphate concen-
tration, promoting the dephosphorylation of Fru-1,6-
bisphosphate in the cytosol (Stitt, 1990), and (2) in the
chloroplast, phosphofructokinase is thought to be
inhibited in the light (Plaxton, 1996). The rationale of
such enzymatic regulations is that hexose molecules
are prevented from entering the glycolytic breakdown,
and so respiration does not consume Suc as soon as it
is synthesized in the cytoplasm.
13C3-Glc, the Fru
Cytoplasmic Glc Contributes to Starch Synthesis
in the Light
When fed with13C-enriched Glc (at 5,500 per mil),
leaves produced
13C-labeled starch (Table II), with
Tcherkez et al.
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a d13C value around 30& higher than without Glc
feeding, indicating that Glc contributed to starch
synthesis. The contribution was nevertheless low,
around only 0.5% of the starch amount (that is,
approximately 0.075 mg mg21fresh weight) was la-
beled (see the ‘‘Result’’ section for calculations). The
flux of starch synthesis from13C-enriched Glc during
the 2 h of feeding treatment in the light was then 0.075
mg mg21/2 h ; 0.038 mg mg21h21; that is, 0.045 mmol
C m22s21, while the total starch synthetic flux
was around 2 mg mg21/3 h ; 0.7 mg mg21h21(Table
II); that is, 0.84 mmol C m22s21. So, Glc fed 0.045/
0.84 ; 5% of starch synthesis in the leaf during the
feeding experiment. The rate of starch synthesis from
13C-enriched Glc is consistent with that found in po-
tato by Quick et al. (1995): chloroplasts extracted from
intact leaves and illuminated with14C-Glc (in a me-
dium containing HCO3
14C-Glc with a rate of approximately 0.04 mmol C
m22s21(recalculated assuming a realistic chlorophyll
amount of 0.4 g Chl m22).
It has also been found that other carbon sources than
photosynthetic CO2can feed starch synthesis in intact
illuminated leaves: Nogue ´s et al. (2004) showed that it
was not possible to completely label starch with CO2
and while the starch amount increased, the d13C value
of starch reached a plateau.
These observations might be paralleled with the
presence of a diffusion-driven Glc phosphate trans-
locator on the inner chloroplast membrane (Scha ¨fer
et al., 1977; Quick et al., 1995), which can feed starch
synthesis with cytoplasmic hexose molecules, al-
though the associated flux appears to be quantitatively
minor in this study.
2) synthesized starch from
Concluding Remarks
To our knowledge, this paper describes the first in
vivo study on the metabolic basis of inhibition by light
of leaf respiration. Clearly, the main inhibited steps are
the entrance of hexose molecules into the glycolytic
pathway and the Krebs cycle. We nevertheless recog-
nize that our experiments were made in typical con-
ditions (21% O2, 350 mL L21CO2, 22?C) and the results
may be influenced by environmental parameters. In-
deed, it has been shown that there lies no inhibition of
respiration by light in some physiological conditions
(Sharp et al., 1984, and refs. therein). Two parameters
are of particular interest: temperature, which is known
to enhance respiratory enzymatic activities, and the
photorespiratory rate (oxygen partial pressure), which
has an effect on the redox status of the mitochondria.
Further experimental data are now needed to investi-
gate the effect of such environmental conditions on
day respiratory metabolism. Moreover, the fact that
the Krebs cycle appears to be slowed down in the light
raises the question of energy in leaf cells and more
precisely, of NADH feeding of the respiratory chain.
Although one may suggest that photorespiration or
the cytoplasmic-mitochondrial malate shuttle have
such a role, further metabolic studies are needed to
determine their respective contribution in vivo.
MATERIALS AND METHODS
Plant Material
French bean (Phaseolus vulgaris) L. cv Contender plants were grown from
seed in 1-L pots of potting mix in a greenhouse, as described by Tcherkez et al.
(2003). Minimum photosynthetic photon flux density during a 16-h photo-
period was maintained at approximately 500 mmol m22s21by supplemental
lighting. Temperature and vapor pressure deficit were maintained at approx-
imately 25.5?C/18.5?C and 1.4/1.2 kPa day/night, respectively. Carbon iso-
tope composition (d13C) of CO2in the greenhouse air was 29.5& 6 0.3&. The
first trifoliar fully expanded leaves were used for all measurements.
Gas Exchange Measurements
Closed System (Dark Respiration)
The respiration chamber was placed in a closed system, which was directly
coupled to an elemental analyzer (EA) NA-1500 (Carlo-Erba, Milan) through
a 15-mL loop, as described by Tcherkez et al. (2003). Briefly, molar fractions of
respiratory CO2were measured with an infrared gas analyzer (IRGA; Finor,
Maihak, Germany) placed in the closed system that was first flushed with
CO2-free air. The loop was shunted when CO2reached around 300 mL L21and
the gas inside the loop was introduced into the EA with helium for gas
chromatography. The connection valve between the elemental analyzer and
the isotope ratio mass spectrometer (VG Optima, Micromass, Villeurbanne,
France) was opened when the CO2peak emerged from the EA.
Open System (Photosynthesis and On-Line Carbon
Isotope Discrimination)
The assimilation chamber was connected in parallel to the sample air hose
of the LI-6400 (LI-COR, Lincoln, NE). This aluminum chamber ([20 3 12 3 6]
1026m3) had a clear Plexiglas lid that allowed us to accommodate the middle
leaflet (typical leaf surface approximately 0.01 m2). Two fans placed in the
chamber gave a boundary layer conductance to water of approximately
6.7 mol m22s21. Leaf temperature was controlled at 20?C with circulating
water from a cooling water bath to the jacket of the leaf chamber and was
measured with a copper-constantan thermocouple plugged to the thermo-
couple sensor connector of the LI-6400 chamber/IRGA. Ingoing air was dried
(at approximately 1 mmol H2O mol21) and passed through the chamber at
a rate of 1 L min21, monitored by the LI-6400. Molar fractions of CO2were
measured with the IRGA of the LI-6400. Light was supplied by a 500-W
halogen lamp (Massive N. V., Kontich, Belgium). The lamp was placed about
30 cm above the chamber and 5 cm of deionized water and 1 cm of glass in the
container filtered the radiation. The photosynthetic photon flux density at leaf
level inside the chamber was maintained at 450 mmol m22s21during the
labeling period. Inlet CO2was obtained from a gas cylinder (Air Liquide,
Grigny, France) with a d13C of 251.2& 6 0.2&. The outlet air of the chamber
was regularly shunted and was sent to the loop to measure its isotope
composition and thus the on-line carbon isotopic discrimination (Dobs). The
gas inside the loop was introduced into the EA for gas chromatography as
described above. Dobsduring photosynthesis was measured following the
method described by Evans et al. (1986; see ‘‘Theory’’).
Day Respiration Measurements
Day respiration was measured with a LICOR-6400 open system, according
to the method described in Peisker and Apel (2001). Briefly, A/cicurves are
done at different light levels and the linear fit gives the CO2compensation
point G and the internal leaf resistance ri. G is plotted as a function of the
product riRnand the slope of the linear regression is m 5 Rd/Rnand so gives
Rd. In bean, the effect of light on the G/rirelationship was slight and did not
modify significantly the estimate of m.
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Starch Extraction Procedure
The extraction procedures for starch were similar to that described by
Tcherkez et al. (2003). Leaf powder was suspended with 1 mL of distilled
water in an Eppendorf tube (Eppendorf Scientific, Hamburg, Germany). After
centrifugation, the pellet was washed four times with 95% ethanol at room
temperature and starch was extracted with HCl solubilization and precipi-
tated with cold methanol. After lyophilization, starch is transferred to tin
capsules (Courtage Analyze Service, Mont Saint-Aignan, France) for isotope
analysis.
NMR
Perchloric acid (PCA) extracts were prepared from 5 g of frozen leaf
material as described by Aubert et al. (1996) for phloem cells. Spectra were
obtained on a spectrometer (AMX 400) equipped with a 10-mm multinuclear
probe tuned at 161.9 and 100.6 MHz for31P- and13C-NMR, respectively. The
deuterium resonance of2H2O (100 mL added per mL of extract) was used as
a lock signal.
Conditions for
intervals and a sweep width of 20 kHz. Broad-band decoupling at 2.5 W
during acquisition and 0.5 W during the delay was applied using the Waltz
sequence; the signal was digitized using 32,000 data points zero-filled to
64,000 and processed with a 0.2-Hz line broadening.13C-NMR spectra are
referenced to hexamethyldisiloxane at 2.7 ppm. Mn21ions were chelated by
the addition of 1 mmol L211,2-cyclohexylenedinitrilotetraacetic acid. The
assignmentsofresonanceof13Cpeakswerecarried outaccordingto Gout etal.
(1993). Identified compounds were quantified from the height of their
resonance peaks using fully relaxed conditions for spectra acquisition (pulses
at 20-s intervals). Peak intensities were normalized to a known amount of the
reference compound (maleate) that is added to the sample (internal standard).
A carbon atom is here considered to be labeled when its estimated positional
13C proportion13C/(13C 112C) is more than 2% (the natural abundance is
nearly 1.1%).
13C-NMR acquisition utilized 19-ms pulses (90?) at 6-s
13C-Enriched Molecules
The positional13C-labeled molecules (99%13C in the considered position)
were purchased to Eurisotop (Saclay, France). Pyruvate was dissolved in
distilled water and pH was corrected to 6.8 with NaOH. To obtain non-fully
labeled solutions, the labeled compounds were mixed with industrial Glc
(d13C 5 29&) or pyruvate (d13C 5 221&) from Sigma. The resulting overall
composition of the Glc and pyruvate solutions was checked to be 5,500& and
2,500&, respectively. In other words, the13C-enriched carbon atom position
had a composition of 47,750& and 8,000&, respectively (the other positions
being at 29& and 221& for Glc and Pyr, respectively). The final concentra-
tion was 0.015 mol L21in all cases. The solutions were poured in an Eppendorf
tube and fed to the leaves through the transpiration stream.
ACKNOWLEDGMENT
Guillaume Tcherkez acknowledges Salvador Nogue ´s for his help for
preliminary works on dark respiration.
Received March 2, 2005; revised April 4, 2005; accepted April 10, 2005;
published June 24, 2005.
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