PhD Thesis proposal form
Discipline: Biology (Plant Ecophysiology)
Doctoral School : ED 145: Plant Sciences / Sciences du Végétal
http://www.ed-sciences-du-vegetal.u-psud.fr/en/ecoledoctorale.htm
Thesis subject title: Impact of (re)fixation of CO2 by PEP-carboxylase on isotopic signature
(13C/12C) of root-respired CO2 (comparison with leaves) under varying nitrogen nutrition.
 Laboratory name and web site: Ecologie, Systématique et Evolution (ESE, CNRS-UMR 8079)
www.ese.u-psud.fr
 PhD supervisor (contact person):
 Name: GHASHGHAIE Jaleh
 Position: Professor, Head of Group “Photosynthesis and Environment” of the
Department of Plant Ecophysiology
 email: Jaleh.ghashghaie@u-psud.fr
 Phone number: ++33(0)1 69 15 63 59
 Thesis proposal (max 1500 words):
Key words : Respiration, Metabolism, Root/leaf, PEPc activity, nitrogen nutrition, carbon stable
isotopes, 13C-labelling.
INTRODUCTION
Isotope discrimination against heavy carbon (13C) occurs during photosynthetic CO2 uptake leading
to a 13C-depletion of plant organic matter (OM), while CO2 left in the atmosphere gets 13C-enriched.
Thus, records of the variation of the isotopic composition of CO2 above ecosystems are generally
used for disentangling photosynthetic and respiratory fluxes by mixing models, assuming that no
post-photosynthetic discrimination occurs. However, the generally accepted hypothesis that no
discrimination does occur downstream the photosynthetic CO2 fixation is now questioned. By
compiling many data from literature we showed that the leaves are in general 13C-depleted compared
to all other organs (Badeck et al. 2005) suggesting that post-photosynthetic discriminations do occur.
Different hypotheses are postulated to explain this difference, e.g. (i) opposite respiratory
fractionation between leaves and heterotrophic organs, (ii) higher PEPc activity (re-fixation of
CO2 by PEP carboxylase via anaplerotic pathway) in heterotrophic organs compared to leaves,
etc. (see review by Cernusak et al. 2009 and references therein). These hypotheses open fields of
experimental studies to investigate the physiological/metabolic mechanisms involved in the
enrichment of heterotrophic organs.
PhD Thesis proposal form
Opposite discrimination during respiration by leaves and roots: Our group investigated the
hypothesis of respiratory fractionation and is the first having demonstrated that the CO2 respired by
leaves in the dark is 13C-enriched compared to leaf organic matter (reviewed by Ghashghaie et al.
2003), while it is 13C-depleted in the case of roots (Bathellier et al. 2008 and references therein).
Although the 13C-enrichment in leaf respired CO2 is now confirmed by different research groups on
many species, there are only a few data concerning the root respiratory fractionation. The opposite
respiratory fractionation between leaves and roots discussed below could partly explain the betweenorgan isotopic differences already demonstrated in many species (see our review Badeck et al, 2005).
We also showed that the isotopic differences between organs (both OM and respired CO2) appeared
only at the beginning of autotrophy acquisition when leaves started to be green (Bathellier et al. 2008).
We had already shown that the isotopic signature of leaf-respired CO2 varied depending on species
(Duranceau et al. 1999; Ghashghaie et al. 2001), environmental conditions (e.g. drought, temperature),
the nature of the substrate used (carbohydrates, lipids or proteins) and on the relative activity of
metabolic pathways (Tcherkez et al. 2003). Interestingly, we showed that isotopic signal of rootrespired CO2 remained unchanged whatever the carbohydrate pool size (Bathellier et al. 2009).
Metabolic origin of opposite fractionation between leaf and root respiration: We proposed that
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C-enrichment in leaf-respired CO2 could be due to the heterogeneous 13C-distribution in hexose
molecules combined with a higher contribution of pyruvate dehydrogenase (PDH) reaction (releasing
13
C-enriched CO2, i.e. C-1 of pyruvate coming from C-3 and C-4 positions of glucose) relative to
Krebs cycle (releasing 13C-depleted CO2, i.e. C-2 and C-3 of pyruvate coming from C-1, C-2, C-5 and
C-6 of glucose) to the overall respiration. This heterogeneous 13C-distribution in hexoses is due to
isotope effect during aldolase reaction enriching C-3 and C-4 positions of hexoses while leaving
behind 13C-depleted trioses (Rossmann et al. 1991). We validated this hypothesis by changing relative
activity of metabolic pathways (PDH vs Krebs cycle) by either increasing leaf temperature or
submitting plants to continuous darkness in Phaesolus and demonstrated that the leaf respired CO2
becomes progressively 13C-depleted in both cases (Tcherkez et al. 2003). We concluded that
glycolysis cannot supply Krebs cycle at high temperature and continuous darkness, resulting in
degradation of reserves e.g. lipids (known to be 13C-depleted) to supply Krebs cycle thus releasing
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C-depleted CO2. A linear relationship obtained between respiratory quotient (RQ) and respired13
CO2 confirmed the metabolic origin of respired-13CO2. Under typical conditions, leaf RQ is around 1
indicating the use of carbohydrates as main substrate for respiration and the respired CO2 is 13Cenriched, while under high temperature and after a long period of darkness, RQ is much lower
indicating the use of proteins or lipids and the respired CO2 is 13C-depleted approaching the isotopic
signature of lipids.
Surprisingly, the root-respired 13CO2 does not follow the leaf-respired 13CO2, and remained
low and almost unchanged despite the decrease in carbohydrate pool size (under continuous darkness)
and despite substantial changes in RQ (substrate switch). Clearly, leaves and roots do behave
differently, presumably because of differences in respiratory metabolic pathways between autotrophic
and heterotrophic tissues. Positional labelling experiments on attached roots of Phaseolus immerged in
glucose or pyruvate solution 13C-labelled in C-1, or C-2 or C-3, allowed estimation of the relative
contribution of PDH, Krebs cycle and pentose phosphate pathway (PPP) to the overall respiration
(Bathellier et al. 2009). Decarboxylation of C-1 of glucose during PPP discriminating against 13C, this
could explain the 13C-depleted CO2 respired by roots. However, the impact of re-fixation of CO2 by
PEPc is not elucidated yet. PEPc discriminating in favour of 13C, this would change the isotopic
signature of both OM and respired CO2.
PhD Thesis proposal form
PROPOSED EXPERIMENT
The general approach is to continue the research program already developed during the previous work
to clarify the meanders of the metabolic functioning of various organs, various plants and under
various environmental conditions. We plan to test the hypothesis of a higher PEPc activity in
heterotrophic organs. We are going to answer these questions: In which extent the (re)fixation of CO2
by PEPc is affected by nitrogen nutrition and in which extent does it affect the isotopic signature of
root OM and root-respired CO2? Labelling experiments using 13C-labelled CO2 will be conducted in a
double-compartment system to label separately photosynthetic assimilation by leaves and anaplerotic
CO2 fixation by roots, as well as to measure the respiratory CO2 of shoot and roots. A device with two
compartments is already built. These experiments will be combined with measurements of 13C content
in metabolites and in respired-CO2 of both organs and will allow to follow the 13C-label fixed by the
photosynthesis and by the PEPc in the respiratory metabolism of each organ, in different pools
supplying root respiration (as we have already done for leaves, see Nogués et al. 2004) and finally to
verify how photosynthesis modifies the signal of the CO2 respired by organs.
Because the rate of PEPc activity is linked to the type of nitrogen assimilation (Lopes & Araus
2006), labelled-CO2 fixation by PEPc will be followed in roots of plants cultivated under different
types of nitrogen nutrition (NO3- or NH4+). We will determine in which extent this nutrition affects the
fixation (incorporation) of the labelled carbon by PEPc in the OM. This work will be realised on a C3
legume (bean, our model plant), a non-legume C3 (sunflower), and 2 plants, one with dominant
reduction of nitrates in roots and the other in leaves (discussion with colleagues is in progress for the
choice of these 2 species).
Overall, this work would allow a better understanding of the origin of the divergence in
isotopic signature between heterotrophic and autotrophic organs, between species and under different
environmental conditions. This study will also provide information at physiological level which can be
up-scaled and compared to the carbon balance studies at ecosystem level of grasslands.
Technical facilities available:
This work will be carried out thanks to the facilities in our laboratory and those at the technical
Platform (Métabolisme-Métabolome) of l’IFR 87 (located at IBP, Orsay), including a Isotope Ratio
Mass Spectrometer (IRMS) coupled to an elemental analyser (EA) allowing isotope analysis of
organic matter (both bulk material and purified metabolites), another EA-IRMS coupled to gas
exchange system allowing on-line measurements of isotope discrimination during photosynthesis and
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CO2-respired by intact leaves or roots. Individual sugars and organic acids are purified using a
HPLC before isotope analysis. Leaf gas exchange will be measured using a portable Licor-6400 for
CO2 and an oxygen electrode for O2. Respiratory quotient (RQ) will be determined as ratio of
respired CO2/O2 consumed. A double-compartment respiratory chamber is already built for isotope
labelling of leaves and roots independently to determine 13CO2 fixation by PEPc. 13C-label in
metabolites will be measured by NMR in Nantes University.
Collaborations foreseen:
Prof. Guillaume Tcherkez (IBP, Orsay) : We have already 16 joint publications in the isotopic
discrimination during respiration from his PhD under my supervision and since his appointment in
Orsay. He is the director of the technical platform at Orsay, where all isotope analyses will be done.
PhD Thesis proposal form
Dr. Franz W. Badeck (PIK-Potsdam, Germany) : We have already 11 joint publications the
isotopic discrimination during respiration, since our collaboration in the frame of European Research
Network (NETCARB). We will continue our 15-year collaboration during this PhD too.
Dr. Richard Robins (University of Nantes) – We started our collaboration for NMR analyses after
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C-labelling.
Prof. Graham Farquhar (ANU, Canberra, Australia) – We have already 3 joint publications on
stable isotopes. We will collaborate for modelling aspect of isotopic data.
Cited references:
Badeck et al. (2005) - Rapid Communications in Mass Spectrometry, 19: 1381-1391.
Bathellier et al. (2008) - New Phytologist, 177: 406-418.
Bathellier et al. (2009) - New Phytologist, 181:387-399.
Duranceau et al. 1999 - Plant, Cell and Environment, 22 :515-523.
Ghashghaie et al. (2001) – Plant, Cell and Environment, 24 : 505-515.
Ghashghaie et al. (2003) – Phytochemistry Reviews, 2: 145-161.
Lopes & Araus (2006) - Physiologia Plantarum, 126, 435-445.
Nogués et al. (2004) - Plant Physiology, 136: 3245-3254.
Rossmann et al. (1991) - Plant Physiology, 96: 609–614.
Tcherkez et al. (2003) - Plant Physiology, 131: 237-244.

Publications of the laboratory in the field (max 5): The names of the supervised students are
underlined.
2010 Tcherkez G., Schäufele R., Nogués S., Piel C., Boom A., Lanigan G., Barbaroux C., Mata C.,
Elhani S., Hemming D., Maguas C., Yakir D., Badeck FW., Griffiths H., Schnyder H. &
Ghashghaie J. – On the 13C/12C isotopic signal of day and night respiration at the mesocosm
level. Plant, Cell & Environment, 33:900-913. IF = 5.145
2009 Bathellier C., Tcherkez G., Mauve C., Bligny R., Gout E. & Ghashghaie J. – On the resilience
of nitrogen assimilation by intact roots under starvation, as revealed by isotopic and
metabolomic techniques. Rapid Communications in Mass Spectrometry, 23 (18): 2847-2856.
IF = 2.846
2009 Bathellier C., Tcherkez G., Bligny R., Gout E., Cornic G. & Ghashghaie J. – Metabolic origin
of d13C of respired CO2 from the roots of Phaseolus vulgaris. New Phytologist, 181:387-399.
IF = 6.516
2005 Badeck F., Tcherkez G., Nogués S., Piel C. & Ghashghaie J. – Post-photosynthetic
fractionation of stable carbon isotopes between plant organs-a widespread phenomenon. Rapid
Communications in Mass Spectrometry, 19: 1381-1391. IF = 2.846 (113 times cited)
2003 Tcherkez G., Noguès S, Bleton J., Cornic G., Badeck F. & Ghashghaie J. – Metabolic origin
of carbon isotope composition of leaf dark-respired CO2 in Phaseolus vulgaris L. Plant
Physiology, 131: 237-244. IF = 6.451 (116 times cited)

Specific requirements to apply, if any:
Knowledge on plant physiology is required.