respiratory carbon fluxes in leaves

Available online at www.sciencedirect.com

Respiratory carbon fluxes in lea
vesGuillaume Tcherkez, Edouard Boex-Fontvieille, Aline Mahe andMichael Hodges
Leaf respiration is a major metabolic process that drives energy

production and growth. Earlier works in this field were focused

on the measurement of respiration rates in relation to

carbohydrate content, photosynthesis, enzymatic activities or

nitrogen content. Recently, several studies have shed light on

the mechanisms describing the regulation of respiration in the

light and in the dark and on associated metabolic flux patterns.

This review will highlight advances made into characterizing

respiratory fluxes and provide a discussion of metabolic

respiration dynamics in relation to important biological

functions.

AddressInstitut de Biologie des Plantes, CNRS UMR 8618, Universite Paris-Sud,

91405 Orsay Cedex, France

Corresponding author: Tcherkez, Guillaume

([email protected])

Current Opinion in Plant Biology 2012, 15:308–314

This review comes from a themed issue on

Physiology and metabolism

Edited by Julian M Hibberd, Andreas PM Weber

Available online 11th January 2012

1369-5266/$ – see front matter

# 2011 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.pbi.2011.12.003

IntroductionRespiration by plant leaves is one of the most important

biochemical processes of carbon exchange between the

vegetation and the atmosphere. CO2 evolution by leaf

respiration is believed to represent �25% of total plant

carbon loss and�3 Gt of CO2 are liberated by leaves each

year from terrestrial ecosystems [1]. Although the mini-

mization of respiratory CO2 loss might be viewed as

desirable to improve plant carbon use efficiency, there

is now compelling evidence that respiration is of crucial

importance to sustain growth and biomass production.

Respiratory metabolism provides intermediates that are

in turn used for nitrogen and sulfur assimilation; wide-

scale studies have shown that there is a positive relation-

ship between leaf respiration and nitrogen content [2].

Since leaf respiration interacts with other metabolic path-

ways like photosynthesis, photorespiration, and nitrogen

assimilation that in turn depend on environmental con-

ditions, respiratory rates can show considerable variations.


In the past few years, much effort has been devoted to

disentangling respiratory fluxes in leaves and the meta-

bolic mechanisms controlling them. In this paper, we

shall summarize recent findings, ongoing mysteries and

integrate them in a tentative metabolic map that

describes leaf respiration in the light and in the dark.

Here, we will focus on respiratory carbon fluxes, that is,

the catabolic pathway by which CO2 is produced via

glycolysis and the tricarboxylic acid pathway (TCAP).

Respiratory fluxes as revealed by13C-fluxomicsFluxomic analyses can be performed in two ways (Figure

1): 13C-labeling (metabolic flux analyses, MFA) or flux-

balance analyses (FBA) inferred from metabolic compo-

sition. Reviews that explain techniques, advantages and

drawbacks of MFA and FBA have already been published

[3,4�]. 13C-based fluxomics take advantage of 13C-label-

ing then returning to 12C (pulse/chase) or isotopic steady

state upon 13C-feeding (Figure 1a). For obvious reasons

(very slow or no turn-over of structural and stored organic

molecules), reaching the isotopic steady state is hardly

feasible in leaves. Also, the natural means by which the13C-label can be provided is limited to 13CO2 with intact,

non-detached leaves and this causes an undesirable build-

up of 13C signals in many metabolites (mainly sugars).

Feeding leaves with 13C-enriched metabolites under

physiological conditions is not straightforward and so

far this has been done through the transpiration stream.

Using these techniques, isotopic tracing with 13C-glucose,13CO2 and 13C-pyruvate has recently provided some

advances in our understanding of respiratory fluxes in

illuminated leaves (Figure 2a): firstly, 13C-glucose mol-

ecules do not enter glycolysis and are committed to

sucrose synthesis [5]; secondly, there is only little con-

sumption by glycolysis of 13C-labeled triose phosphates

synthesized by photosynthesis from 13CO2 [6��]; thirdly,

positional 13C-labeling with pyruvate has shown an

imbalance between the decarboxylation by pyruvate

dehydrogenase (PDH) and the TCAP [5,7]. That is,

decarboxylation of 13C-pyruvate by the TCAP in mostly

inhibited (up to 95% in Phaseolus vulgaris and 80% in

Xanthium strumarium) while the PDH decarboxylating

activity is decreased by ca. 30% in the light compared

to the dark [5,6��]. The well-recognized inhibition of the

mitochondrial PDH activity is caused by phosphorylation

[8] and perhaps, the accumulation of NH3 and acetyl-CoA

that non-competitively downregulate PDH catalysis [9]

(but note that the chloroplastic PDH is not downregu-

lated in the light). The inhibition of TCAP-catalyzed

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Respiratory carbon fluxes Tcherkez et al. 309

Figure 1

(a)

(b)

Labeling (e.g., 13C1-Glc)

Isotopic analysisNMR

Positional 13C- enrichmentin metabolites

Sucrose

Fructose

Pyruvate

Malate

etc.

Selection of metabolicreactions

Assumed metabolicpathways

Set of equations describingpositional 13C-compositions

Calculation of metabolic fluxes thatsatisfactorily explain the observed

isotope distribution

Prediction of 13C-enrichment in CO2

Isotopic analysisIRMS

13C-enrichment inrespired CO2

Hypotheses on the metabolicorigin of respired CO2

Match andvalidation

Constraint(s)(e.g. normalizing

fluxes to the input)

List of known reactions orgenes encoding enzymes

Other outputs (e.g., total ATPor NADPH demand and

production)

Calculation of metabolic fluxes thatsatisfactorily explain the metabolic

composition

Constraints(e.g. carbon source,

growth rate, etc.)

Assumed metabolic pathways

Metabolic composition

Mass-balance equationsdescribing metabolic contents

Computed CO2 and O2respiratory fluxes and

respiratory quotient (RQ)Experimental gas exchange Match and

validation

Current Opinion in Plant Biology

Comparison of metabolic flux analysis (MFA) (a) and genome-scale flux-balance analysis (FBA) (b). For each, the simplified procedure is depicted

(black) and a possible way to validate or confirm calculated respiratory flux patterns is indicated (blue). As an example, MFA is illustrated with 13C-1-

glucose labeling (labeled C-atom in white) and the subsequent isotopic redistribution. FBA takes advantage of the precise and quantitative metabolic

composition (colored pie chart) to infer the probable flux pattern from which it originates.

13CO2 decarboxylation in the light is likely due to: firstly,

the feedback inhibition by NADPH and NADH and other

effectors [10], secondly, the abstraction of C-skeletons for

N and S assimilation from the TCAP and thirdly, the

remobilization of 12C-reserves (e.g. stored citrate) [11��].Accordingly, when leaves are labeled with 13CO2 in the

light, calculated 13C-commitments into respiratory


intermediates indicate that the TCAP is not a cycle in

the light [6��]: citrate is neosynthesized from acetyl-CoA

and oxaloacetate at a rather slow rate and therefore, citrate

utilization is divorced from malate metabolism. In other

words, citrate conversion to 2-oxoglutarate (2OG) and

glutamate seems to operate independently of oxaloacetate

production by the phosphoenolpyruvate carboxylase


310 Physiology and metabolism

Figure 2

Pyruvate

Acetyl-CoA

Citrate

Isocitrate

2-oxoglutarate

Succinate

Oxaloacetate

Malate

Fumarate

Glu

Gln Glu

NH3 NO2-

NO3-

Phosphoenolpyruvate

Triose-P

HCO3- HCO3

-

Glc-6-P

UDP-Glc

Suc

NADPH NADP

P-GY 3-P-GA

oxaloacetate malate

phosphoenolpyruvate pyruvate

acetyl-CoAFatty acids, etc.CO2

CO2

CO2 CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2

Reserves

Recycling?

Pentosephosphates

cPDH

mPDH

mCS

3GAPDHENO

mMDH

PEPC

SDH

2OGDHSCoL

ACO

FUM

cMDH

GABA

Succinic-semialdehyde

GABP

PPDKPK

Reserves

CO2

Photosynthesis

Ala

chloroplast

mitochondrion

(a)Triose-P

Pyruvate

Acetyl-CoA

Citrate

Isocitrate

2-oxoglutarate

Succinate

Oxaloacetate

Malate

Fumarate Glu

Phosphoenolpyruvate

Starch Glc-6-P, Malt Glc-6-P

UDP-Glc

Suc

phosphoenolpyruvate pyruvate

acetyl-CoAFatty acids, etc.

Reserves

Pentosephosphates

Glc-6-P, Malt

Pentosephosphates

Asp

GlnAsn

cPDH

3GAPDHENO

PK

mPDH

mCS

mMDH

PEPC

SDH 2OGDHSCoL

ACO

FUM

ME

chloroplast

mitochondrion

(b)

Reserves

Day respiratory fluxes Light-to-dark transition and dark respiratory fluxes

Current Opinion in Plant Biology

Simplified scheme of respiratory fluxes showing the enzymatic steps discussed in the text. The flux associated with the malic enzyme during LEDR is

indicated with a pink arrow. Broken and thin-grey arrows stand for multiple steps and low fluxes, respectively. For clarity, this scheme does not

represent the interaction with photorespiration nor the O2-uptake by the mitochondrial electron chain. The involvement of cytoplasmic isoforms is not

represented. Abbreviations: ACO, aconitase; ENO, enolase; cMDH, chloroplastic malate dehydrogenase; cPDH, chloroplastic pyruvate

dehydrogenase; FUM, fumarase; GABA, g-aminobutyrate; GABP, GABA permease; mCS, mitochondrial citrate synthase; ME, malic enzyme; mMDH,

mitochondrial malate dehydrogenase; mPDH, mitochondrial pyruvate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase; PK, pyruvate kinase;

PPDK, pyruvate Pi dikinase; P-GY, 3-phosphoglycerate; SCoL, succinyl-CoA ligase; SDH, succinate dehydrogenase; 2OGDH, 2-oxoglutarate

dehydrogenase; 3GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 3-P-GA, 3-phosphoglyceraldehyde.

(PEPC) which instead, feeds malate and fumarate pools.

This pattern of organic acid metabolism further accords to

recent findings on the light-enhanced dark respiration

(LEDR), defined as the transitory increase in rate of release

of CO2 by leaves just after transfer from light to dark.

This peak in leaf respiration is believed to come from the

decarboxylation of malate because the decline of the

malate content parallels CO2 evolution by LEDR (Figure

2b) [12]. In addition, both LEDR-evolved CO2 and malate

are naturally 13C-enriched [12,13�]. The relationship be-

tween the LEDR and the inhibition of respiration in the

light is further demonstrated by the close correlation

between the degree of light inhibition of day respiration

(CO2 evolution) and the difference in respiration rates

between the LEDR peak and the steady-state dark respir-

ation rate [14]. That is, the inhibition of key respiratory

enzymes and the non-cyclic nature of the TCAP in the

light allow the build-up of malate and fumarate pools,

which are rapidly decarboxylated during LEDR when

leaves are darkened. Such a situation is believed to differ

markedly in the dark where the TCAP has been known for

years to operate at a high rate as a proper cycle [15,16].


Mostly, the light/dark regulation of leaf respiration stems

from biochemical control (metabolic effectors, feedback,

and post-translational modifications) (reviewed in the

introduction of [6��]); however, a few diel changes in

protein abundance (e.g. citrate synthase and aconitase)

have recently been demonstrated within the mitochondrial

proteome [17�].

FBA is not well suited to leaf metabolism per se due to

intensive exchange between leaves and other plant parts

that alter leaf metabolic composition (Figure 1b). That is,

the metabolic composition of leaves does not reflect only

intrinsic leaf metabolic fluxes but also gain and losses

from/to other organs [4�]. Genome-scale FBA that

mimicked leaf metabolism were attempted recently

[18]. However, these analyses failed to give a faithful

representation of leaf metabolism: as the biomass growth

rate was fixed to a certain value, computed fluxes through

the Calvin cycle and the Rubisco-catalyzed carboxylation

increased when photorespiration (O2-fixation) was con-

sidered — in contrast to the actual experimental response

to low CO2. Furthermore, this also caused a lower flux



through the TCAP, suggesting that in illuminated leaves,

photorespiration indirectly (i.e. by mass-balance) partici-

pates to inhibiting day respiratory metabolism.

Respiratory fluxes as revealed by mutationsOver the past few years, key enzymatic mutants have

been generated and their metabolic phenotypes analyzed.

In particular, mutants in which one metabolic step is

reduced or disrupted do give clues on alternative meta-

bolic routes or on adjustments of commitments at branch-

ing points. Mutants affected in cytoplasmic glycolysis at

the level of 3-phosphoglyceraldehyde dehydrogenase

(3GAPDH) [19] and enolase (ENO) [20] do not show a

consistent respiratory phenotype: the dark respiration rate

is affected in the latter and not in the former, and the

changes in organic acid content (e.g. isocitrate) are in

opposite directions. Such contrasted effects are probably

due to the thermodynamic status and the redox role of the

reactions considered. 3GAPDH-catalyzed equilibrium

favors 1,3-bisphosphoglycerate + NADH over 3-phos-

phoglyceraldehyde + Pi + NAD and thereby contributes

to providing NADH and ATP. A visible effect on the

respiration rate is thus expected. ENO is associated with a

well-reversible reaction that involves neither NAD nor

ATP. Mutants affected in the TCAP show very different

metabolic phenotypes depending on the step considered.

Unfortunately, there are quite a few pleitropic effects on

photosynthesis. For example, fumarase [21] and succinyl-

CoA ligase [22] mutants have lower photosynthesis rates

due to a lower stomatal conductance and photosynthetic

capacity, respectively. In the double mutant affected in

two genes encoding NAD-dependent malate dehydro-

genase (MDH), photosynthesis is also lower compared to

the wild type but unexpectedly, so is photorespiration (as

suggested by a smaller post-illumination CO2 burst and a

lower Warburg effect) [23]. Still, the respiration rate (CO2

evolution) is higher both in the light and in the dark,

maybe providing evidence for a higher activity of the

malic enzyme (ME). That is, the absence of metabolic

flux that interconverts malate into oxaloacetate may be

compensated for by the degradation of malate into pyr-

uvate by the ME (with, maybe, a futile cycle involving

PEPC and ME). Thus MDH and ME activities seem

crucial to accommodate changes in the TCAP flux. In a

mutant affected in mitochondrial citrate synthase (mCS),

excess oxaloacetate is directed to malate, aspartate, and

asparagine accumulation and excess phosphoenolpyruvate

is directed to aromatic amino acids [24]. In addition,

mutants affected in ME have generally less organic acids

(significantly less citrate) and more sugars or amino acids

such as glutamate and aspartate in the dark [25]. A further

comparison of metabolic changes caused by mutations has

been carried out to calculate control coefficients of dark

respiration (relative change of the flux of interest with

respect to the enzymatic step considered as measured by

its in vitro activity) [26]. Although crude (since it does not

account for alternative CO2 production or concerted


changes in other enzymatic activities), this approach

indicates that dark respiration CO2-flux is controlled by

aconitase and 2OG dehydrogenase. Uncertain control

coefficients are obtained for mCS and fumarase (either

close to zero or very negative) as well as MDH (large

positive or negative values). Jacobian-based mathemat-

ical simulations of the metabolic system that minimized

partial derivatives further suggested that the respiratory

pathway cannot reach stability when the sensitivity of

individual fluxes to metabolite content was uniform [27].

A steady state may be achieved with feedback inhibition

by oxaloacetate and pyruvate on MDH and ME, respect-

ively. Taken as a whole, MDH and ME activities have

thus proved essential for respiratory control.

TCAP alternative pathwaysThere is now good evidence that alternative respiratory

pathways (‘bypasses’) occur in leaves (Figure 2b). First, the

PEPC, that synthesizes oxaloacetate from phosphoenolpyr-

uvate, is believed to be of crucial importance to feed the

TCAP (anaplerotic role) in the light from which intermedi-

ates are abstracted by, for example, ammonium assimila-

tion. This PEPC-flux has been demonstrated using carbon

isotopes, by observing naturally 13C-enriched malate and

aspartate [28] and 13C-fractionation during photosynthesis

[29]. The metabolic flux associated with the PEPC is quite

large, probably near 5% of net photosynthesis. Second, the

ME, that converts malate into pyruvate + CO2, has been

shown to be minimal in the light compared to the dark [30]

but it is responsible for the LEDR peak (see above). Third,

the use of lysine degradation products to sustain the

respiratory electron chain has been suggested to occur in

mutants affected in succinate dehydrogenase (respiratory

complex II) [31]. Fourth, the g-aminobutyric acid shunt

(GABA-shunt) seems to accompany the TCAP [6] and may

compensate for a reduced succinyl-CoA ligase activity [31].

Furthermore, changes in respiratory metabolism in

mutants affected in GABA transporters (permease)

strongly suggest the involvement of the GABA-shunt aside

the TCAP [32��]. This is probably related to alanine

metabolism since the imbalance between pyruvate syn-

thesis (by pyruvate kinase) and pyruvate consumption (by

the TCAP) would be alleviated by a diversion to alanine,

catalyzed by alanine–succinate semialdehyde transamin-

ase. Fifth, excess pyruvate may also be converted back to

phosphoenolpyruvate by the pyruvate phosphate dikinase

in the light, thereby facilitating the PEPC-catalyzed reac-

tion [33] and perhaps, inorganic phosphate (Pi) and pyr-

ophosphate homeostasis. In heterotrophic plant cells,

changes in cytoplasmic glucose-6-phosphate-to-Pi ratio

immediately triggers a decrease in both cytoplasmic pH

and cell respiration [34]. The rationale of these multiple

alternative pathways is presently not clear but possibly,

they represent metabolic strategies compensating for the

inhibition of the TCAP in the light while interacting

metabolisms (nitrogen assimilation and photorespiration)

are intense.



Interactions with other metabolismsRespiratory metabolism provides 2OG to the Gln synthe-

tase/Glu synthase cycle that converts 2OG into Glu, one

key step of nitrogen integration onto organic molecules. It

is believed that leaf nitrogen reduction and assimilation

mostly occurs in the light and therefore the role of day

respiration should be critical. As described above, leaf

respiration is inhibited by light and so the origin of the

carbon atoms used to synthesize 2OG is probably not

from recent photosynthetic assimilates but rather,

reserves of organic acids. Recently, double 13CO2 and15N-NH4NO3 labeling of rapeseed leaves (Brassica napus)has shown that most neosynthesized 15N-containing mol-

ecules inherit C-atoms from reserves and not from current

photosynthesis [11��]. These observations agree with the

very slow isotopic turn-over of Glu when Arabidopsisrosettes are labeled with 13CO2 and returned back to12CO2 [35]. Non-reciprocally, most 13C-amino acids are

also 15N-labeled upon double labeling, showing the

importance of nitrogen assimilation as a privileged fate

for neosynthesized 2OG molecules [11]. It should be

noted that these conclusions are certainly valid for sulfur

assimilation, since very little 13C-labeling is seen in

methionine and cysteine, which are the main products

of S reduction and assimilation.

Nitrogen primary metabolism also represents the link

between day respiration and photorespiration. In fact,

the measurement of 13C-pyruvate decarboxylation by

the TCAP under varying CO2/O2 ratios have shown that

both TCAP activity and Glu neosynthesis are promoted

under high photorespiratory conditions on a short-term

basis (within two hours) in cocklebur leaves (X. strumarium)

[7]. Similarly, rather old experiments on spinach proto-

plasts (Spinacia oleracea) showed a larger 14C-commitment

into Glu and Gln at low [CO2] [36]. The measurement of

the chloroplastic electron flow to nitrite reduction further

suggests that nitrogen assimilation is favored when plants

are grown at low [CO2] in several species [37]. However, a

metabolic model based on steady state, mass-balance

equations applied to all metabolites indicates that low

photorespiration rates should decrease day respiration

and TCAP activity [38]. There is therefore no present

consensus on the relationship between photorespiration

and day respiration. It is nevertheless likely that day

respiration is simultaneously influenced by changes in

nitrogen availability, photorespiration and photosynthesis

and acclimates when such changes are permanent.

Recently, growth under high [CO2] has been shown to

be accompanied by an increase in both respiration rate and

abundance of transcripts encoding enzymes throughout

the respiratory pathway [39�].

Leaf respiration can also be studied in terms of O2-uptake

flux or the respiratory quotient (RQ, i.e. the ratio of CO2-

release to O2-uptake). Simultaneous measurements of

CO2 and O2 fluxes during night respiration in intact leaves


have already been carried out (the average RQ value is 1

under typical conditions) and indicate some changes with

environmental conditions such as leaf temperature. By

contrast, respiratory O2-consumption in the light is poorly

documented: it has been shown to be either very small

[40] or similar to that in the dark [41]. As a result, the RQ

of day respiration is currently unknown. On the one hand,

the production of NADH (or reducing equivalents,

malate) in the light by photosynthesis and photorespira-

tion is believed to sustain the electron respiratory chain,

causing a quite large O2-consumption rate. On the other

hand, the rate of CO2 evolution in the light is smaller than

that in the dark due to metabolic downregulations. Thus

presumably, the RQ of illuminated leaves is small (<1). If

true, this would not indicate that the respiratory substrate

has a low elemental oxygen content (like, e.g. lipids) but

rather, that O2-uptake and CO2 production are only

loosely metabolically linked. Such a pattern is never-

theless likely to be affected by environmental conditions

such as nitrogen nutrition. Electron flow measurements

have suggested that upon nitrate supply, the O2-con-

sumption flux by mitochondria in the light is lower due

to the competition between nitrate reduction and respir-

atory dehydrogenases (of the mitochondrial electron

chain) for NAD(P)H [42].

ConclusionsThe recent work summarized here has improved our

understanding of leaf respiratory fluxes by showing clear

day/night metabolic dynamics and complex interactions

with C and N assimilations. Nevertheless, much remains

to be done to better characterize the influence of environ-

mental conditions on day respiration. Up to now, tech-

nical difficulties have impeded scientific progress on this

topic — while the body of data on dark respiration is now

considerable. New laser-based techniques that allow day

respiration to be carefully followed with isotopes (either

at natural abundance or after labeling) with a high time-

resolution [13�] should provide significant advances in

respiratory carbon sources and respiratory dynamics

(light-to-dark and dark-to-light transitions) in the near

future.

AcknowledgementsThe authors thank the Agence Nationale de la Recherche for its support througha grant Jeunes Chercheurs, under contract no. JC08-330055. G.T. thanks Prof.Graham Farquhar, Dr. Josette Masle and Dr. Margaret Barbour forproviding assistance during the time of writing this paper. The financialsupport of the Institut Universitaire de France is acknowledged.

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Michaeli S, Fait A, Lagor K, Nunes-Nesi A, Grillich N, Yellin A,Bar D, Khan M, Fernie AR, Turano FJ, Fromm H: A mitochondrialGABA permease connects the GABA shunt and the TCA cycle,and is essential for normal carbon metabolism. Plant J 2011,67:485-498.


http://dx.doi.org/10.1111/j.1365-3040.2011.02332.x


This paper demonstrates that AtBABP is a mitochondrial GABA per-mease and describes the physiological effects of the associated muta-tion. The use of 14C-GABA clearly shows the involvement of mitochondrialGABA import to sustain the TCAP.

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General transcriptomic analyses were carried out to investigate the effectof growth under high [CO2] in soybean leaves. A clear increase oftranscript abundance associated with TCAP enzymes was demon-strated, which accords with the previous study from the same lab thatshowed an increased number of mitochondria per cell under high [CO2].

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http://dx.doi.org/10.1104/pp.111.180711

respiratory carbon fluxes in leaves

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