Review
Tansley review
Opposite carbon isotope discrimination during
dark respiration in leaves versus roots – a review
Author for correspondence:
Jaleh Ghashghaie
Tel: +33(0)1 69 15 63 59
Email: [email protected]
Jaleh Ghashghaie1 and Franz W. Badeck2,3
Received: 2 June 2013
Accepted: 15 September 2013
Protaso, 302, 29017, Fiorenzuola d’Arda (PC), Italy; 3Potsdam Institute for Climate Impact Research (PIK), PF 60 12 03, 14412,
1
Laboratoire d’Ecologie, Systematique et Evolution (ESE), CNRS UMR8079, B^atiment 362, Universite de Paris-Sud (XI), F-91405,
Orsay Cedex, France; 2Consiglio per la Ricerca e la sperimentazione in Agricoltura, Genomics research centre (CRA - GPG), Via San
Potsdam, Germany
Contents
Summary
751
I.
Introduction
751
II.
Photosynthetic carbon isotope discrimination
752
III.
Post-photosynthetic discrimination
753
IV.
Apparent dark respiratory fractionation in leaves
754
13
V.
Root-respired CO2 is generally C-depleted compared with
root organic matter, except in C3 woody plants
757
VI.
d13CR of leaves and roots diverges at leaf autotrophy onset
762
VII.
Metabolic pathways potentially implied in 13C depletion
in root-respired CO2
763
VIII.
A glance at stem respiration
765
IX.
Impact on carbon isotope composition of plant
organic matter
765
Conclusions
766
Acknowledgements
766
References
766
X.
Summary
New Phytologist (2014) 201: 751–769
doi: 10.1111/nph.12563
Key words: anaplerotic pathway, C3
herbaceous vs woody species, carbon isotope
discrimination, leaves vs roots, metabolic
pathways, ontogeny, pentose phosphate
pathway, respiration.
In general, leaves are 13C-depleted compared with all other organs (e.g. roots, stem/trunk and
fruits). Different hypotheses are formulated in the literature to explain this difference. One of
these states that CO2 respired by leaves in the dark is 13C-enriched compared with leaf organic
matter, while it is 13C-depleted in the case of root respiration. The opposite respiratory
fractionation between leaves and roots was invoked as an explanation for the widespread
between-organ isotopic differences. After summarizing the basics of photosynthetic and postphotosynthetic discrimination, we mainly review the recent findings on the isotopic composition
of CO2 respired by leaves (autotrophic organs) and roots (heterotrophic organs) compared with
respective plant material (i.e. apparent respiratory fractionation) as well as its metabolic origin.
The potential impact of such fractionation on the isotopic signal of organic matter (OM) is
discussed. Some perspectives for future studies are also proposed .
I. Introduction
Until recently, changes in the 13C signal of ecosystem-respired CO2
have been attributed to the changes in photosynthetic discrimination caused by changes in environmental conditions (Fung et al.,
1997). However, the generally accepted hypothesis that no
discrimination occurs downstream of photosynthetic CO2 fixation
is now in question. As leaves are generally 13C-depleted compared
with all other organs (e.g. roots, stem/trunk and fruits) in C3 plants,
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it is suggested that post-photosynthetic discriminations do probably occur, leading to the observed isotopic difference between
autotrophic and heterotrophic tissues/organs. Different hypotheses
are formulated in the literature to explain this difference. Several
studies have shown that CO2 respired by leaves in the dark is 13Cenriched compared with leaf organic matter (see Supporting
Information, Notes S1, for references used for Fig. 1(a); and for a
review see Ghashghaie et al., 2003; Werner & Gessler, 2011), while
it is 13C-depleted in the case of root respiration (Badeck et al., 2005;
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Klumpp et al., 2005; Schnyder & Lattanzi, 2005; Bathellier et al.,
2008; Gessler et al., 2009; Wegener et al., 2010; Kodama et al.,
2011; Zhu & Cheng, 2011). The opposite respiratory fractionation
between leaves and roots discussed in the following could partly
explain the widespread between-organ isotopic differences
(reviewed by Badeck et al., 2005; Cernusak et al., 2009). Although
the 13C enrichment in leaf-respired CO2 has now been confirmed
by different research groups for many C3 species (reviewed by
Ghashghaie et al., 2003; Werner & Gessler, 2011), data concerning
root respiratory fractionation are scarce (see review by Werth &
Kuzyakov, 2010).
In this paper, after summarizing the basics of photosynthetic and
post-photosynthetic discrimination, we mainly review the recent
findings on the isotopic composition of CO2 respired (d13CR) by
leaves (autotrophic organs) and roots (heterotrophic organs)
compared with respective plant material (i.e. apparent respiratory
fractionation) as well as its metabolic origin. We also briefly
describe apparent fractionation during stem respiration. The
potential impact of such fractionation on the isotopic signal of
organic matter (OM) is also discussed, and the effect of difference in
the presumable source of respired organic material is assessed. Some
perspectives for future studies are also proposed.
70
(a) Leaves
C4 herbs
C3 woody
C3 herbs
No fractionation
60
50
40
30
20
10
0
Number of observations
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(b) Roots
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20
15
10
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14
(c) Stems
12
10
II. Photosynthetic carbon isotope discrimination
8
Discrimination against the heavy carbon isotope 13C occurs
during photosynthetic CO2 uptake, leading to 13C depletion in
plant OM compared with atmospheric CO2. Photosynthetic
discrimination is, on average, c. 20& in C3 and c. 4& in C4
leaves. It has been extensively studied and robust models have
been developed for both plant types (Farquhar et al., 1982;
Farquhar, 1983). The simple version of the C3 model is based on
the two main discriminating steps: discrimination during CO2
diffusion from the ambient air into the leaves through stomata
(4.4&) and during carboxylation by ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco, 29&). Although the complete
version of the model includes leaf internal resistance to CO2
diffusion and discrimination during day respiration and photorespiration, these aspects are less well investigated (for a review, see
Brugnoli & Farquhar, 2000). In some C3 species, leaf mesophyll
resistance has been shown to be high enough to lead to a relevant
decrease in the CO2 mole fraction in the chloroplasts (relative to
the intercellular air spaces), thus decreasing net photosynthetic
discrimination compared with the values expected from the
simple model (Evans et al., 1986, 2009; von Caemmerer & Evans,
1991). Photorespiratory fractionation has been shown to be up to
12& against 13C, thus blurring the on-line photosynthetic
discrimination measurements (Lanigan et al., 2008). The impact
of day respiratory fractionation on the net photosynthetic
discrimination is difficult to determine, because the mitochondrial respiration, mainly the tricarboxylic acid cycle (TCA or
Krebs cycle), is strongly inhibited in the light (Tcherkez et al.,
2005). Recently, day-respired CO2 has been shown to be 13Cdepleted by 0–10& compared with C3 leaf OM (Tcherkez et al.,
2011). However, the simple version of Farquhar’s model has been
validated for many C3 species and is often used rather than the
6
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4
2
0
–16 –14 –12 –10 –8
–6
ΔR (‰) =
–4
δ13C
–2
0
–
δ13C
S
2
4
6
8
R
Fig. 1 (a, b) Distribution of different classes of apparent respiratory
fractionation values (DR) of leaves (a) and roots (b) of C3 herbs (dark gray
bars), C3 woody plants (light gray bars), and C4 herbs (black bars) under
varying growth conditions and measured in the dark using different
methods. (c) A few data on stem respiration of five woody C3 species are also
presented. References used for leaf data from 23 herbaceous C3, 20 woody
C3 (including six coniferous) and four herbaceous C4 species are listed in
Supporting Information Notes S1. For roots, data from 12 herbaceous C3
species, eight woody C3 species (including two coniferous) and five
herbaceous C4 species listed in Table 1 are used. DR is calculated as the
difference between the carbon isotope composition of leaf or root material
(d13CS) available in the literature (bulk organic matter, water-soluble fraction
or soluble sugars, as respiratory substrates) and that of leaf- or root-respired
CO2 (d13CR) as a product of respiration. Negative DR values correspond to
13
C enrichment and positive DR values to 13C depletion in respired CO2
compared with the substrate. Vertical dashed lines indicate no respiratory
fractionation (i.e. DR = 0).
complete version, owing to its complexity, to determine internal
resistances and photorespiratory discrimination in parallel to
Ci : Ca.
According to the simple version of Farquhar’s model, the overall
discrimination during photosynthetic assimilation of CO2 in C3
leaves is a linear function of the intercellular to ambient CO2 mole
fraction ratio (i.e. Ci : Ca). Changes in environmental conditions
(e.g. air humidity, light intensity, CO2 concentration) affecting
stomatal conductance, and thus Ci : Ca, lead to changes in
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photosynthetic discrimination in C3 leaves (C4 discrimination is
much less dependent on Ci : Ca; for a recent review, see Cernusak
et al., 2013). Changes in internal leaf conductance and photosynthetic capacities also affect Ci : Ca through modification of the sink
strength for CO2. Thus, carbon isotope composition of plant OM
is, in general, considered to properly reflect the carbon isotope
discrimination of a given photosynthesizing system during
photosynthetic CO2 assimilation. However, the carbon isotope
signature of plant OM not only integrates discrimination processes
during net CO2 assimilation (i.e. photosynthesis, photorespiration
and day respiration, already included in the complete version of
discrimination model), but also post-photosynthetic processes,
which could potentially discriminate, for example, night respiration and photoassimilate export from source leaves to sink tissues
(for a recent review, see Cernusak et al., 2009). Post-photosynthetic
discrimination could explain the observed widespread interorgan
isotopic differences (Badeck et al., 2005) as well as the changes in
leaf OM isotopic composition compared with recent photoassimilates. For instance, night respiration of C3 leaves was shown to
release, in general, 13C-enriched CO2 compared with photosynthetic assimilates (reviewed by Ghashghaie et al., 2003; Werner &
Gessler, 2011). Such fractionation is expected to change the carbon
isotope signature of the remaining leaf material at the end of the
night period compared with the photosynthetic products fixed
during the daytime (Ghashghaie et al., 2003).
Another consequence of photosynthetic discrimination is that
the CO2 left in the atmosphere becomes 13C-enriched. This effect is
used for retrieving the distribution of sources and sinks from the
spatiotemporal distribution of carbon isotopes in the atmosphere
(Fung et al., 1997), as well as for estimating the isotopic signature of
ecosystem-respired CO2 using Keeling plots and disentangling the
relative contribution of photosynthetic and respiratory fluxes to the
net ecosystem exchanges (Pataki et al., 2003; Mortazavi et al.,
2006). For these studies, the carbon isotope signal of ecosystemrespired CO2 was assumed to reflect the carbon isotope signature of
photosynthetic products and changes in photosynthetic discrimination resulting from changing environmental conditions. However, the hypothesis that no discrimination occurs after net
photosynthetic CO2 fixation in the leaves has been refuted in
recent years (Ghashghaie et al., 2003; Ogee et al., 2003; Badeck
et al., 2005; Bowling et al., 2008). There is now growing interest in
post-photosynthetic discrimination as well as its impact on the
isotope composition of both plant OM and CO2 respired by
different plant organs and ecosystem components (for recent
reviews, see Bowling et al., 2008; Br€
uggemann et al., 2011; Werner
et al., 2012), because of the potential impacts on predicted
isofluxes, additional constraints on spatiotemporal variation in
the component fluxes determining the integrated isotopic composition of the ecosystem-respired CO2 and the attribution of
respiratory CO2 to its carbon sources.
III. Post-photosynthetic discrimination
Carbon isotope composition of leaf bulk OM is often used as a
reference for photosynthetic discrimination in both plant- and
ecosystem-level studies. Badeck et al. (2005) compiled data from
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the literature on c. 80 plant species, including C3 and C4,
herbaceous and woody, as well as annual and perennial plants. They
clearly showed that the 13C signal in OM varies between organs in
C3 plants, the leaves in most cases being 13C-depleted compared
with all other organs: only slightly compared with green stems, c.
1& in average compared with roots and c. 2& compared with tree
trunks. A difference in carbon isotope composition is also observed
between different tissues of a given organ; for example, leaf lamina
were shown to be 13C-depleted compared with leaf ribs in different
herbs and woody species (Badeck et al., 2009 and references
therein). Heterotrophic leaves (before photosynthetic onset) are
13
C-enriched compared with green autotrophic leaves as well
(Bathellier et al., 2008; Lamade et al., 2009). It is concluded that
fractionation mechanisms do probably occur after net CO2 fixation
in the leaves, leading to the observed 13C differences between
autotrophic and heterotrophic tissues/organs. Indeed, in the event
that no fractionation occurs downstream of photosynthetic CO2
fixation in the leaves, no between-organ isotopic difference can be
expected, because of the conservation law. Various hypotheses
about post-photosynthetic discrimination have already been
advanced to explain the isotopic differences between autotrophic
and heterotrophic organs/tissues – in the main these are as follows:
(1) opposite respiratory fractionation between leaves and heterotrophic organs (Badeck et al., 2005; Klumpp et al., 2005; Bathellier
et al., 2008);
(2) export of isotopically heavier assimilates from leaves to sink
organs as a result of fractionation during phloem loading/transport
(Hobbie & Werner, 2004; Gessler et al., 2007) and/or developmental variation in photosynthetic discrimination with lower Ci :
Ca in mature leaves thus exporting 13C-enriched assimilates to sink
organs (Francey et al., 1985; Cernusak et al., 2001);
(3) higher rate of CO2 fixation by phosphoenolpyruvate carboxylase (PEPc) in heterotrophic tissues/organs compared with C3
leaves (Terwilliger & Huang, 1996);
(4) differential use of 13C-depleted day vs 13C-enriched night
sucrose between leaves and sink tissues (Tcherkez et al., 2004;
Gessler et al., 2008);
(5) emission of volatile organic compounds (VOCs) and ablation
of surface waxes, both of which are, in general, 13C-depleted
compared with photosynthetic products;
(6) discrimination during root exudation (Badeck et al., 2005);
(7) use of assimilates produced under contrasting environmental
conditions and thus contrasting in d13C for asynchronous growth
of different organs (Pate & Arthur, 1998; Cernusak et al., 2002,
2005).
Cernusak et al. (2009) recently reviewed the hypotheses explaining why leaves are generally 13C-depleted compared with all other
organs.
The basis of these processes is fractionation that occurs at
metabolic branching points mainly during C–C bond making or
cleavage, resulting in a nonuniform intramolecular 13C distribution as well as between-metabolite isotopic differences. As a
consequence, when a given molecule with heterogeneous 13C
distribution is cleaved during a given enzymatic reaction, a
so-called ‘fragmentation fractionation’ (Tcherkez et al., 2004)
occurs, resulting in new molecules carrying the positional 13C
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signal of the fraction of the educt molecule that makes up the
individual product. For instance, heterogeneous 13C distribution
within molecules could (in addition to enzymatic isotope effects)
result in 13C-enriched or 13C-depleted CO2 evolved during
decarboxylation of such molecules, releasing heavy or light carbon
atom positions and leaving behind 13C-depleted or 13C-enriched
molecules, respectively.
Already within the Calvin cycle in the chloroplasts (daytime),
aldolase, which condenses two trioses to form fructose 1-6
bisphosphate, discriminates in favour of 13C, enriching C-3 and
C-4 positions of hexose molecules (Rossmann et al., 1991; Gleixner
& Schmidt, 1997; Gilbert et al., 2011, 2012), thus enriching
transitory starch in 13C, while 13C-depleted trioses left behind are
transported to the cytosol, forming sucrose. Consequently, day
sucrose is expected to be 13C-depleted relative to the average of all
assimilates, while night sucrose coming from degradation of
transitory starch will be 13C-enriched. Thus, a differential use of
13
C-depleted day sucrose vs 13C-enriched night sucrose is expected
to impact on the observed between-organ isotopic differences (see
Hypothesis 4 earlier and the model of Tcherkez et al. (2004), as well
as experimental evidence in Gessler et al. (2008)). As the fractionation by aldolase already occurs in the chloroplasts, the term ‘postcarboxylation’ fractionation was suggested by Gessler et al. (2008)
for fractionation after carboxylation by Rubisco in the chloroplasts.
The term post-photosynthetic discrimination is generally used for
processes after net CO2 fixation and sugar synthesis in the leaves.
Amongst post-photosynthetic processes that are potentially
discriminating, it is primarily respiration that has been investigated
during the past decade. By contrast with photosynthetic discrimination, respiratory discrimination is complex, not only because
different pathways involving different enzymatic decarboxylations
contribute to respiration (i.e. various substrates can be used), but
also because the isotope effects of the enzymes involved, and
consequently the 13C signature of CO2 evolved, can change
depending on the relative commitment of the substrates at
metabolic branching points to decarboxylations and to other
reactions. Moreover, if more than one substrate with different
isotopic signature is involved in feeding respiration, the source
carbon used and its isotopic composition cannot be accurately
determined. For all these reasons, the term ‘apparent’ respiratory
fractionation (denoted DR) is generally used (Ghashghaie et al.,
2003) to describe the isotopic difference between bulk OM taken as
source carbon (or putative substrates, mainly sugars) and the
product (overall released CO2).
As phloem sugars are often 13C-enriched compared with leaf
sugars, fractionation during phloem loading and transport of
assimilates (Hypothesis 2) was suggested (Gessler et al., 2007) and
theoretical models were proposed (Hobbie & Werner, 2004;
Werner & Gessler, 2011). However, the gradients in sugar isotope
composition along the tree trunks reported in the literature are
contradictory, that is, there is either 13C enrichment or 13C
depletion from the top to the bottom (Keitel et al., 2003; Gessler
et al., 2004, 2007; Scartazza et al., 2004; Werner et al., 2012). The
effects of other post-photosynthetic fractionation processes, such
as, for example, ablation of leaf waxes, emission of VOCs and
anaplerotic reactions, on the isotopic composition of OM have not
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yet been experimentally quantified, except for the pioneering work
by Nalborczyk (1978) discussed later.
IV. Apparent dark respiratory fractionation in leaves
The carbon isotopic composition of plant-respired CO2 has long
been considered to be similar to plant OM. However, pioneering
investigations in the early 1970s showed some variability in carbon
isotope composition of dark-respired CO2 by leaves compared with
leaf material and thus in ‘apparent’ respiratory isotope fractionation
(reviewed by Ghashghaie et al., 2003). Because of this variability
and difficulties in identifying substrates used for respiration, this
topic was not investigated further for nearly 25 yr. Carbon isotope
composition of plant-respired CO2 has long been considered to be
equal to that of photosynthetic products, thereby reflecting
photosynthetic fractionation only. However, experimental data
published during the past decade have clearly demonstrated not
only that a nonnegligible ‘apparent’ dark respiratory fractionation
occurs in C3 leaves, but also that it is highly variable, confirming the
pioneering data. Although CO2 respired in the dark by C3 leaves is
shown to be generally 13C-enriched compared with leaf OM (or
leaf sugars) under typical conditions, it substantially changes (13C
enrichment up to 15& or 13C depletion in some cases) among
species and with environmental conditions (see reviews by
Ghashghaie et al., 2003; Badeck et al., 2005; Werner et al.,
2012), showing marked diel dynamics depending on functional
groups as well (see the recent review by Werner & Gessler, 2011,
and references therein). Published data clearly show this large
variability between species and plant types (Fig. 1). Nevertheless,
respiratory CO2 of C3 herbs and C3 woody plants is, on average,
more 13C-enriched than that of C4 plants (P = 0.0023 and
P = 0.0095, respectively, Wilcoxon test). However, the 13C
enrichment in respired CO2 is not significantly different between
C3 herbs and woody plants (P = 0.0625). Leaf-respired CO2 of C3
species (both herbaceous and woody plants) is generally
13
C-enriched. A few exceptions showing 13C-depleted respired
CO2 correspond, in the case of herbaceous species, to respiration of
young seedlings of sunflower and peanut (Smith, 1971), and thus
13
C depletion in respired CO2 may result from the use of lipids as
substrate; and, in the case of woody species, to very young Quercus
ilex (Werner et al., 2009), probably because the respiration was
measured at the end of the night period (and so carbohydrate
reserves were exhausted and lipids were used as substrates), and to
some coniferous trees (Troughton et al., 1974; Mortazavi et al.,
2005). The nature of the organic material used as the presumable
source (OM vs water-soluble or a carbohydrate fraction) for the
determination of apparent discrimination does not impact on these
main findings. In none of the 41 cases of C3 herbaceous leaves for
which the isotopic signature of OM as well as water-soluble organic
matter (WSOM) or a carbohydrate fraction was reported did the
sign of the apparent discrimination differ between calculation on an
OM basis or a WSOM/carbohydrate basis. For these cases, OM
was, on average, 2.25& more negative than WSOM or the
carbohydrates. Thus, apparent fractionation calculated on a
WSOM or carbohydrate basis qualitatively matches the results
on an OM basis, but leads to a lower estimate of discrimination.
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C3 herbs
C3 woody plants
5
ΔR (‰) = δ13CCH – δ13CR
5
13
0
13
C depletion
13
C depletion
13
C enrichment
0
C enrichment
–5
–5
–10
–10
Leaves
1 : 1 relationship
Roots
No fractionation
–15
–15
–10
–5
0
Leaves
1 : 1 relationship
Roots
No fractionation
5
–15
–10
–5
0
–15
5
ΔR (‰) = δ13COM – δ13CR
Fig. 2 Apparent respiratory fractionation (DR) calculated with measurements of carbohydrates (CH; sucrose or starch) or the water-soluble fraction (WSOM) as
the putative substrate vs apparent respiratory fractionation calculated with organic matter (OM) as the reference for C3 herbs (left panel) and woody C3 plants
(right panel). Open symbols, leaf respiration; closed symbols, root respiration. Solid lines correspond to a 1 : 1 relationship and dashed lines indicate no
respiratory fractionation (i.e. DR = 0). Positive values (indicated by white arrows) correspond to 13C depletion and negative values (indicated by black arrows) to
13
C enrichment in respired CO2 compared with plant material used as putative substrate. Literature data used are those from Fig. 1 for which both OM and CH
or WSOM were available.
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–16
Data from literature on different species (leaves)
Bean leaves (Tcherkez et al., 2003)
Regression (leaves)
Bean roots (Bathellier et al., 2009)
Regression (roots)
–18
–20
δ13CR (‰)
Among 36 cases of C3 leaves of woody species for which the isotopic
signature of OM as well as WSOM or a carbohydrate fraction was
reported, only three differed in the sign of the apparent discrimination between calculation on an OM basis and calculation on a
WSOM/carbohydrate basis. OM was on average 1.04& less
negative (i.e. 13C-enriched) than WSOM or the carbohydrates.
The three exceptions with changing sign of the respiratory
discrimination stem from measurements on Prosopis velutina (see
Fig. 2).
The observed 13C enrichment in dark-respired CO2 by C3 leaves
was initially proposed (Ghashghaie et al., 2001) to be primarily the
result of the heterogeneous 13C distribution in hexose molecules
mentioned earlier, combined with a higher contribution of
pyruvate dehydrogenase reaction, PDH (releasing 13C-enriched
CO2 (i.e. C-1 of pyruvate coming from C-3 and C-4 positions of
glucose)), relative to TCA 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. Tcherkez et al. (2003) obtained
a positive linear relationship (Fig. 3) between leaf d13CR and
respiratory quotient (RQ = CO2 evolved/O2 consumed), demonstrating that when glycolysis could not supply carbon skeletons
needed for the TCA cycle (because of the increase in respiration rate
with increasing temperature, or because of the decrease in
carbohydrate pool size under prolonged darkness), reserves such
as fatty acids (known to be 13C-depleted) are oxidized to replete the
TCA cycle, thereby releasing 13C-depleted CO2. Under typical
dark conditions, the leaf RQ is c. 1, indicating the use of
carbohydrates as the main substrate for respiration and that the
respired CO2 is 13C-enriched, while under high temperatures and
after a long period of darkness, the RQ is much lower, indicating
the switch to the use of less oxygenated substrates, such as fatty
acids, and that the respired CO2 is 13C-depleted (c. 30&),
approaching the isotopic signature of lipids (Fig. 3, open circles;
–22
–24
–26
–28
–30
–32
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
RQ
Fig. 3 Variations of carbon isotope composition of CO2 d13CR respired by
attached leaves (open symbols) and washed attached roots (closed symbols)
of Phaseolus vulgaris as a function of respiratory quotient (RQ = CO2
evolved/O2 consumed). Regression lines are also presented. Data points
correspond to individual measurements on different plants. Leaf data are
from Tcherkez et al. (2003) and root data are from Bathellier et al. (2009).
Additional data extracted from the literature for several other plant species
(tomato, castor bean, peanut, pea and radish) are indicated by stars (James,
1953; Park & Epstein, 1961; Smith, 1971). Redrawn from Ghashghaie &
Tcherkez. 2013. Advances in botanical research, chap. 8, vol. 67, Ó 2013,
with permission from Elsevier.
Fig. 4, right-side panel). According to the intramolecular 13Cdistribution values for hexose molecules given by Rossmann et al.
(1991), leading to a fragmentation fractionation, leaf d13CR is
expected to vary between c. 21& (if only PDH contributes to
respiration) and c. 27& (if the TCA cycle is the sole contributor),
suggesting that the variability in metabolic pathways could lead to
the observed variability in leaf d13CR (Tcherkez et al., 2003). The
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LEAVES
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1200
(a)
(d)
1000
30
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25
20
600
15
400
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200
5
(b)
1.2
(e)
R (nmol CO2 m–2 s–1)
R (nmol CO2 g–1 DW s–1)
ROOTS
0
RQ
1.0
0.8
0.6
(c)
–22
(f)
δ13CR (‰)
–24
–26
Carbohydrates
Carbohydrates
–28
Proteins
Proteins
–30
Lipids
–32
–34
Lipids
0
1
2
3
4
5
6
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Time in darkness (d)
variability in the isotopic signature of leaf-respired CO2 could thus
potentially be used as an indicator of metabolic pathways and
substrates used for respiration (see reviews by Ghashghaie et al.,
2003; Werner & Gessler, 2011; Ghashghaie & Tcherkez, 2013).
However, the flux balance between PDH and the TCA cycle
alone cannot explain the measured variations in leaf d13CR, ranging
between 15& and 33&, observed for different species under
varying conditions (Tcherkez et al., 2003; Barbour et al., 2007;
Priault et al., 2009; Werner et al., 2009; Wegener et al., 2010),
because they exceed the estimated range mainly for the 13Cenriched values. Isotope effects operating by PDH and decarboxylating enzymes involved in the TCA cycle could also influence the
overall d13CR. Moreover, as pyruvate is at a metabolic branching
point, the fractionation by PDH will depend on the relative
commitment of pyruvate to decarboxylation or other reactions.
Therefore, the leaf d13CR and its variability reflect the concerted
influence of metabolic pathways and fluxes, isotope effects of the
enzymes involved, as well as the intermolecular 13C distribution in
hexoses and the resulting fragmentation fractionation.
In addition, new techniques, mainly tunable diode laser
spectroscopy (TDLS; Bowling et al., 2003), allowing high timeNew Phytologist (2014) 201: 751–769
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3
4
Fig. 4 (a–f) Variations of respiration rate, R
(a, d), respiratory quotient, RQ (b, c) and
carbon isotope composition of CO2 respired
(c, f) by washed attached roots (a–c) and
attached leaves (d–f) of Phaseolus vulgaris
during continuous darkness. (c, f) Dashed
horizontal lines indicate the mean values of
carbon isotope composition of carbohydrates
(soluble sugars), soluble proteins and total
lipids extracted from roots (c) and leaves (f).
Closed circles, data published in Bathellier
(2008); open circles, data from Tcherkez
et al. (2003). Error bars correspond to SE
(n = 3). The leaf mass area index of the first
adult leaves at this developmental stage was
c. 28 2.25 g DW m 2 (n = 3). The leaf
respiration rate on mass basis was thus
between c. 7 and 31 nmol g 1 DW s 1.
resolution measurements during light-to-dark transition, demonstrated strong 13C enrichment in leaf-respired CO2 up to 11&
compared with phloem sap sugars in Ricinus (i.e. d13CR was nearly
15&; Barbour et al., 2007). This result was confirmed using
rapid in-tube sampling of respired CO2 on other C3 species (Priault
et al., 2009; Werner et al., 2009; Wegener et al., 2010). Such strong
13
C enrichment in d13CR of illuminated leaves immediately after
transfer to the dark cannot be explained by decarboxylation of the
13
C-enriched carbon atom position of pyruvate during the PDH
reaction (expected to be c. 21& only) alone, but rather is
attributed to a rapid consumption of 13C-enriched malate
accumulated during the light period (Barbour et al., 2007; Gessler
et al., 2009; Werner et al., 2011). Because of the inhibition of
mitochondrial respiration in the light (Tcherkez et al., 2005),
organic acids such as malate (or oxaloacetate) accumulate in the
cytosol during the light period and are subsequently decarboxylated
by malic enzyme in the mitochondria upon transfer to darkness,
resulting in a peak in the respiration rate lasting 5–20 min, the
so-called light-enhanced dark respiration (LEDR). In C3 plants,
malate and oxaloacetate are indeed 13C-enriched metabolites
compared with sugars, because they originate from anaplerotic
Ó 2013 The Authors
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fixation of HCO3 by PEPc. Because hydration of CO2 by
carbonic anhydrase discriminates in favour of 13C, HCO3 is
enriched in 13C by c. 9& compared with CO2, and PEPc
discriminates only by c. 2.2& against 13C; thus malate issued
from the anaplerotic pathway, and thereby CO2 evolved by
its decarboxylation upon transfer to darkness (LEDR), are
13
C-enriched.
To our knowledge, the impact of respiratory discrimination on
the 13C signature of plant OM has not yet been investigated in a
systematic manner. A 13C depletion by c. 1& in the leaf OM
remaining at the end of the night period compared with
photosynthetic products was estimated using a simple mass balance
calculation and assuming an average value of 4& dark respiratory
fractionation (Ghashghaie et al., 2003). Thus, the 13C-enriched
leaf-respired CO2 during the night could partly explain the
generally observed 13C depletion in leaf organic material compared
with other organs (for a recent review, see Cernusak et al., 2009).
Reports in the literature on the carbon isotope fractionation
during respiration by C4 leaves and by organs other than leaves (e.g.
tree trunks/twigs, roots) are still scarce. A few pioneering data on C4
plants (Smith, 1971; Troughton et al., 1974) and preliminary data
on maize leaves (Ghashghaie et al., 2003) show a small difference
(< 2&) between respired CO2 and bulk OM or leaf sugars, the
difference being either negative or positive (see Fig. 1a). Higher
(re)fixation of respired CO2 by PEPc in C4 leaves compared with
C3 leaves could be at the origin of the low ‘apparent’ respiratory
fractionation observed in C4 leaves, but investigations at the
metabolic level are needed to confirm this hypothesis. One
exception is the high 13C enrichment (10.5&) in respired CO2
compared with leaf OM observed for Paspalum dilatatum cultured
in sand at 15°C (Schnyder & Lattanzi, 2005).
V. Root-respired CO2 is generally 13C-depleted
compared with root OM, except in C3 woody plants
In contrast to leaves, root respiratory fractionation is poorly
investigated, mainly because of difficulties in disentangling the
relative contribution of roots and soil microorganisms (Grossiord
et al., 2012). Understanding of root respiratory fractionation is
necessary, in particular because root respiration is a major
contributor to soil CO2 efflux and thus is an important component
of ecosystem respiration. Root d13CR can be determined under
controlled conditions either on excavated and cleaned/washed
roots (or root samples), that is, roots without soil and without
microorganisms (except for roots including mycorrhizas), or on a
simplified culture system (e.g. hydroponic medium or sand/
vermiculite). Otherwise, in the case of in situ respiration measurements, the soil-respired CO2 carries the isotopic signature of both
roots and microorganisms of the rhizosphere and thus labelling
experiments are needed to distinguish the contribution of each
component.
The isotope composition of root-respired CO2 of herbaceous
C3 plants (alfalfa, perennial ryegrass and sunflower) cultured in
pots filled with sand showed that, in contrast to shoots, rootrespired CO2 was 13C-depleted (up to 5.6& for ryegrass)
compared with root OM but varied with species and with growth
Ó 2013 The Authors
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Review 757
conditions (Klumpp et al., 2005; Schnyder & Lattanzi, 2005).
Similarly, a 13C depletion by c. 1–3& in root-respired CO2
compared with root organic material (or root sucrose) was
observed on washed roots (without soil) of bean plants, both
attached and detached (Badeck et al., 2005; Bathellier et al.,
2008). Whatever the culture support used (peat soil, sand or
vermiculite), the respired CO2 of washed roots had the same
isotope signature, c. 28& for bean plants (Bathellier, 2008). So
far, published data show that, similar to leaves, root d13CR is also
variable within species and conditions (Table 1). On average,
apparent root respiratory fractionation did not differ between C3
and C4 herbs (P > 0.1, Wilcoxon test), while it was significantly
different for roots of C3 woody plants compared with C3 as well as
C4 herbs (P < 0.001). Root-respired CO2 is 13C-depleted compared with root material in herbaceous C3 species, with two
exceptions, Melissa officinalis (Wegener et al., 2010) and Triticum
aestivum (Kodama et al., 2011), surprisingly showing
13
C-enriched root-respired CO2 at the end of the night period,
perhaps because the carbohydrate reserves were exhausted and
lipids were used as substrates. In contrast to C3 herbs, deciduous
C3 trees (Eucalyptus, Acer and Acacia) and shrubs (Halimium
halimifolium and Rosmarinus officinalis) have 13C-enriched rootrespired CO2. For instance, 13C enrichment in root d13CR is
> 9& for Acer negundo (Moyes et al., 2010) and for a Mediterranean semideciduous shrub, H. halimifolium (Dubbert et al.,
2012), except when the latter is cultured in hydroponic medium
(Wegener et al., 2010). This contrast in root d13CR values between
herbaceous and woody species is not observed for leaves, which
show 13C enrichment in respired CO2 for both herbaceous and
woody C3 species (see Fig. 1b), with few exceptions in both plant
types. Since in trees (and shrubs) the root respiration includes the
respiration of associated mycorrhizas (even in cleaned or washed
roots), this could explain the opposite root d13CR values between
trees and herbaceous plants. However, d13CR values should be
measured on mycorrhizas alone to confirm this hypothesis.
Further experiments in the field and in the pots of the same species
should also be conducted to examine a potential effect of field vs
potted conditions. Another hypothesis can be built on the
assumption that, in lignified roots of woody species, C4-like
assimilation of CO2 may occur (J. Bloemen, pers. comm.), that is,
high activity of C4 enzymes and subsequent decarboxylation of
13
C-enriched C4 metabolites such as malate, similar to the high C4
activity observed on C3 stems and petioles (Hibberd & Quick,
2002) and tree twigs (Berveiller & Damesin, 2008). However, as
we do not expect diurnal changes in fixation and release of C4
compounds in roots, this hypothesis requires additional assumptions on variation in time of the rates of assimilation and release;
alternatively, it may be an artefact of the release of C4 compounds
subsequent to extraction of roots from their natural environment.
Structural and metabolic studies on roots of woody species should
be undertaken in order to examine this hypothesis.
Surprisingly, in the case of coniferous trees, root d13CR is similar
to root OM or even slightly 13C-depleted (Dijkstra & Cheng,
2007; Epron et al., 2011). However, the literature on coniferous
tree roots is scarce and more data are needed to confirm this result. It
is worth emphasising the point that CO2 respired by needles of
New Phytologist (2014) 201: 751–769
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Tansley review
Table 1 Carbon isotope composition of root material analysed (d13COM) and root-respired CO2 (d13CR) as well as root apparent respiratory fractionation (DR)
calculated as DR = d13COM – d13CR
d13COM
(&)
DR (&)
Root
material
Culture conditions
Methods for d13CR
Sources
–
–
+ 2.95
OM
Sand + nutrients
Klumpp et al. (2005)
High light pretreatment
–
–
+ 1.52
OM
Sand + nutrients
High light/high nitrogen
–
–
+ 3.73
OM
Sand + nutrients
High light/low nitrogen
–
–
+ 2.84
OM
Sand + nutrients
Low light/high nitrogen
–
–
+ 2.39
OM
Sand + nutrients
Low light/low nitrogen
–
–
+ 2.68
OM
Sand + nutrients
–
–
+ 5.39
OM
Sand + nutrients
24.9
21.7
+ 3.2
OM
Sand + nutrients
28.3
22.8
+ 5.5
OM
Sand + nutrients
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
20.65
+ 2.61
WSOM
Sand + nutrients
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
Klumpp et al. (2005)
Species and conditions
d13CR
(&)
C3 plants (herbs)
Medicago sativa L. (alfalfa)
Low light pretreatment
Lolium perenne L.
(perennial ryegrass)
25°C/23°C
15°C/14°C
Helianthus annuus L. (sunflower)
Low density
23.26
Low density
23.26
22.79
+ 0.46
OM
Sand + nutrients
High density
24.81
21.44
+ 3.36
WSOM
Sand + nutrients
High density
24.81
22.81
+ 2.0
OM
Sand + nutrients
DAS 40–42
28.97
28.24
+ 0.73
OM
DAS 60–62
29.77
28.46
+ 1.31
OM
Sand : perlite : soil
(50 : 9 : 1)
Sand : perlite : soil
(50 : 9 : 1)
29.2
27.6
+ 1.6
Sucrose
Peat soil + nutrients
DAS 22
29.2
28.2
+ 1.0
OM
Peat soil + nutrients
DAS 22
29.4
26.2
+ 3.2
Sucrose
Vermiculite + nutrients
DAS 22
29.4
27.8
+ 1.6
OM
Vermiculite + nutrients
26.5
+ 0.95
Sucrose
Vermiculite + nutrients
29.3
+ 2.2
WSOM
Sand + nutrients
Phaseolus vulgaris L. (bean)
DAS 22
Mean of a 6-d dark
27.45
period
Ricinus communis L. (castor bean)
Daily mean values
31.5
Daily mean values
31.5
28.7
+ 2.8
OM
Sand + nutrients
Morning
32.5
30.4
+ 2.1
WSOM
Sand + nutrients
Morning
32.5
29.3
+ 3.2
OM
Sand + nutrients
Evening
30.5
28.2
+ 2.3
WSOM
Sand + nutrients
Evening
30.5
28.1
+ 2.4
OM
Sand + nutrients
New Phytologist (2014) 201: 751–769
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Klumpp et al. (2005)
Klumpp et al. (2005)
Klumpp et al. (2005)
Klumpp et al. (2005)
Klumpp et al. (2005)
Klumpp et al. (2005)
Schnyder & Lattanzi
(2005)
Schnyder & Lattanzi
(2005)
Klumpp et al. (2005)
Klumpp et al. (2005)
Klumpp et al. (2005)
Zhu & Cheng (2011)
Zhu & Cheng (2011)
On-line d13CR on
attached washed roots
On-line d13CR on
attached washed roots
On-line d13CR on
attached washed roots
On-line d13CR on
attached washed roots
On-line d13CR on
detached washed roots
Badeck et al. (2005)
Keeling plots on excised
cleaned roots
Keeling plots on excised
cleaned roots
Keeling plots on excised
cleaned roots
Keeling plots on excised
cleaned roots
Keeling plots on excised
cleaned roots
Keeling plots on excised
cleaned roots
Gessler et al. (2009)
Badeck et al. (2005)
Bathellier et al.
(2008)
Bathellier et al.
(2008)
Bathellier et al.
(2009)
Gessler et al. (2009)
Gessler et al. (2009)
Gessler et al. (2009)
Gessler et al. (2009)
Gessler et al. (2009)
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Review 759
Table 1 (Continued)
Species and conditions
Glycine max L. (soybean)
DAS 40–42
d13CR
(&)
d13COM
(&)
DR (&)
Root
material
Culture conditions
Methods for d13CR
Sources
Sand : perlite : soil
(50 : 9 : 1)
Sand : perlite : soil
(50 : 9 : 1)
Sand
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
Keeling plots on excised
cleaned roots
Keeling plots on excised
cleaned roots
Keeling plots on excised
cleaned roots
Zhu & Cheng (2011)
In-tube d13CR on excised
root tips
In-tube d13CR on excised
root tips
In-tube d13CR on excised
roots
In-tube d13CR on excised
roots
Wegener et al.
(2010)
Wegener et al.
(2010)
Wegener et al.
(2010)
Wegener et al.
(2010)
In-tube d13CR on excised
root tips
In-tube d13CR on excised
root tips
Wegener et al.
(2010)
Wegener et al.
(2010)
In-tube d13CR on excised
root tips
In-tube d13CR on excised
root tips
Wegener et al.
(2010)
Wegener et al.
(2010)
In-tube d13CR on excised
roots
In-tube d13CR on excised
roots
J. Ghashghaie et al.
(unpublished)
J. Ghashghaie et al.
(unpublished)
29.66
27.87
+ 1.79
OM
29.35
27.73
+ 1.62
OM
27.3
27.1
+ 0.20
OM
27.28
26.48
+ 0.80
OM
DAS 60–62
28.85
27.91
+ 0.94
OM
Daily mean values
25.05
24.18
+ 0.87
WSOM
End of the night
22.7
24.2
1.50
WSOM
End of the day
28.1
24.7
+ 3.40
WSOM
Melissa officinalis L.
End of the night
24.6
27.31
2.71
WSOM
Hydroculture
End of the day
28.0
27.13
+ 0.87
WSOM
Hydroculture
28.7
28.1
+ 0.6
WSOM
Hydroculture
28.7
28.5
+ 0.2
OM
Hydroculture
25.6
24.87
+ 0.73
WSOM
Hydroculture
27.5
24.94
+ 2.56
WSOM
Hydroculture
28.6
28.78
+ 0.5
WSOM
Hydroculture
29.3
26.82
+ 1.2
WSOM
Hydroculture
30.83
29.75
+ 1.08
WSOM
Vermiculite + nutrients
30.83
31.35
0.52
OM
Vermiculite + nutrients
Solanum tuberosum L. (potato)
Tuber (day 23)
27.88
27.09
+ 0.79
OM
Germination in the dark of
tubers in air (without soil)
On-line d13CR on excised
sprouts
On-line d13CR on excised
sprouts
On-line d13CR on excised
sprouts
On-line d13CR on excised
sprouts
On-line d13CR on excised
sprouts
On-line d13CR on excised
sprouts
Maunoury-Danger
et al. (2009)
Maunoury-Danger
et al. (2009)
Maunoury-Danger
et al. (2009)
Maunoury-Danger
et al. (2009)
Maunoury-Danger
et al. (2009)
Maunoury-Danger
et al. (2009)
DAS 60–62
Triticum aestivum L.
(wheat)
DAS 40–42
Mean of all root parts
(18:00)
Mean of all root parts
(18:00)
Salvia officinalis L.
End of the night
End of the day
Oxalis triangularis A.St.-Hil.
End of the night
End of the day
Arachis hypgaea (peanut)
Stage 14 (BBCH)
Stage 14 (BBCH)
Sand : perlite : soil
(50 : 9 : 1)
Sand : perlite : soil
(50 : 9 : 1)
Field (sandy-loamy silt
soil)
Field (sandy-loamy silt
soil)
Field (sandy-loamy silt
soil)
Zhu & Cheng (2011)
Cheng (1996)
Zhu & Cheng (2011)
Zhu & Cheng (2011)
Kodama et al. (2011)
Kodama et al. (2011)
Kodama et al. (2011)
Sprout (day 23)
27.42
25.43
+ 1.99
OM
Tuber (day 23)
27.88
26.06
+ 1.82
Starch
Sprout (day 23)
27.42
23.80
+ 3.62
Starch
Tuber (day 23)
27.88
26.33
+ 1.55
Sucrose
Sprout (day 23)
27.42
24.61
+ 2.81
Sucrose
C3 woody species (trees & shrubs)
Acer negundo
17.9
27.2
9.3
OM
Mature trees
d13CR of soil & roots – soil
without root
Moyes et al. (2010)
Eucalyptus delegatensis L.
March 2005
24.86
26.78
1.92
WSOM
Mature trees
Gessler et al. (2007)
24.86
28.03
3.17
OM
Mature trees
Keeling plots on excised
cleaned roots
Keeling plots on excised
cleaned roots
March 2005
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
Gessler et al. (2007)
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760 Review
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Tansley review
Table 1 (Continued)
Species and conditions
d13CR
(&)
d13COM
(&)
DR (&)
Root
material
Culture conditions
Methods for d13CR
Sources
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
Sun et al. (2012)
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
Dubbert et al. (2012)
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
Dubbert et al. (2012)
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
Dubbert et al. (2012)
In-tube d13CR on excised
root tips
In-tube d13CR on excised
root tips
Wegener et al.
(2010)
Wegener et al.
(2010)
Prosopis velutina
Pre-monsoon
22.2
26.4
4.2
OM
Field sampling
Pre-monsoon
22.2
24.3
2.1
Sucrose
Field sampling
Monsoon
23
25.7
2.7
OM
Field sampling
Monsoon
23
23.9
0.9
Sucrose
Field sampling
23.9
25.7
1.8
WSOM
Open site trees
May
23.9
27.5
3.6
OM
Open site trees
August
19.0
26.8
7.8
WSOM
Open site trees
August
19.0
26.4
7.4
OM
Open site trees
May
23.6
26.2
2.6
WSOM
Forest site trees
May
23.6
27.3
3.7
OM
Forest site trees
August
22.5
28.1
5.6
WSOM
Forest site trees
August
22.5
27.3
4.8
OM
Forest site trees
22.8
25.9
3.1
WSOM
Open site
May
22.8
27.8
5.0
OM
Open site
August
21.4
25.7
4.3
WSOM
Open site
May
24.3
26.6
2.3
WSOM
Forest site
May
24.3
26.9
2.6
OM
Forest site
August
20.4
28.0
7.6
WSOM
Forest site
August
20.4
27.7
7.3
OM
Forest site
23.4
26.3
2.9
WSOM
Open site
May
23.4
27.0
3.6
OM
Open site
August
17.4
25.4
8.0
WSOM
Open site
May
24.1
27.8
3.7
WSOM
Forest site
May
24.1
27.7
3.6
OM
Forest site
August
18.5
27.9
9.4
WSOM
Forest site
August
18.5
27.5
9.0
OM
Forest site
26.1
23.8
+ 2.3
WSOM
Hydroculture
27.3
24.8
+ 2.5
WSOM
Hydroculture
Acacia longifolia L.
May
Rosmarinus officinalis L.
May
Halimium halimifolium L.
May
Halimium halimifolium L.
End of the night
End of the day
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Sun et al. (2012)
Sun et al. (2012)
Sun et al. (2012)
M. Dubbert et al.
(unpublished)
Dubbert et al. (2012)
M. Dubbert et al.
(unpublished)
Dubbert et al. (2012)
M. Dubbert et al.
(unpublished)
Dubbert et al. (2012)
M. Dubbert et al.
(unpublished)
M. Dubbert et al.
(unpublished)
Dubbert et al. (2012)
Dubbert et al. (2012)
M. Dubbert et al.
(unpublished)
Dubbert et al. (2012)
M. Dubbert et al.
(unpublished)
M. Dubbert et al.
(unpublished)
Dubbert et al. (2012)
Dubbert et al. (2012)
M. Dubbert et al.
(unpublished)
Dubbert et al. (2012)
M. Dubbert et al.
(unpublished)
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
New
Phytologist
Tansley review
Review 761
Table 1 (Continued)
Species and conditions
d13CR
(&)
d13COM
(&)
DR (&)
Root
material
Culture conditions
Methods for d13CR
Sources
In-tube d13CR on excised
roots
In-tube d13CR on excised
roots
Photoassimilate labelling
Wegener et al.
(2010)
Wegener et al.
(2010)
Dijkstra & Cheng
(2007)
Epron et al. (2011)
Mean of all root parts
(18:00)
Mean of all root parts
(18:00)
Pinus ponderosa
32.2
30.1
+ 2.1
WSOM
Hydroculture
32.2
29.8
+ 2.4
OM
Hydroculture
–
–
OM
Field
Pinus pinaster
27.31
0.4
to + 1.5
+ 0.69
OM
Field (sandy podsol)
In-tube d13CR on excised
washed roots
On-line d13CR on
attached roots in sand
On-line d13CR on
attached roots in sand
Schnyder & Lattanzi
(2005)
Schnyder & Lattanzi
(2005)
Werth & Kuzyakov
(2005)
Werth & Kuzyakov
(2005)
Werth & Kuzyakov
(2005)
Zhu & Cheng (2011)
C4 plants
Paspalum dilatatum
25°C/23°C
26.62
–
–
+ 0.6
OM
Sand + nutrients
–
–
+ 5.9
OM
Sand + nutrients
15.8
15.1
+ 0.7
OM
Hydroculture
0.1 9 Full nutrient
solution
Deionised water
14.6
14.8
–0.2
OM
Hydroculture
14.2
14.5
–0.3
OM
Hydroculture
DAS 40–42
16.90
13.93
+ 2.97
OM
DAS 60–62
18.49
13.98
+ 4.51
OM
Sand : perlite : soil
(50 : 9 : 1)
Sand : perlite : soil
(50 : 9 : 1)
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
20.71
13.64
+ 7.07
OM
13.90
+ 7.01
OM
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
Zhu & Cheng (2011)
20.91
Sand : perlite : soil
(50 : 9 : 1)
Sand : perlite : soil
(50 : 9 : 1)
20.14
13.41
+ 6.73
OM
13.41
+ 6.19
OM
CO2 trapping by NaOH,
attached roots
CO2 trapping by NaOH,
attached roots
Zhu & Cheng (2011)
19.60
Sand : perlite : soil
(50 : 9 : 1)
Sand : perlite : soil
(50 : 9 : 1)
14.7
13.9
+ 0.8
OM
Field sampling
Sun et al. (2012)
Pre-monsoon
14.7
13.8
+ 0.9
Sucrose
Field sampling
Monsoon
17.3
13.5
+ 3.8
OM
Field sampling
Monsoon
17.3
16.5
+ 0.8
Sucrose
Field sampling
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
In-tube d13CR on excised
cleaned roots
15°C/14°C
Zea mays L. (maize)
Full nutrient solution
Amaranthus tricolor L.
DAS 40–42
DAS 60–62
Sorghum bicolor L.
DAS 40–42
DAS 60–62
Sporobolus wrightii
Pre-monsoon
Zhu & Cheng (2011)
Zhu & Cheng (2011)
Zhu & Cheng (2011)
Sun et al. (2012)
Sun et al. (2012)
Sun et al. (2012)
DAS, d after sowing; WSOM, water-soluble fraction extracted from root material.
Positive DR values correspond to 13C depletion and negative DR values to 13C enrichment in respired CO2 compared with root material analysed. Growth
conditions and methods for respired CO2 collection for isotope analysis are indicated. C3 woody species (trees and shrubs) are presented separately from the C3
herbs. The coniferous species are indicated by grey shading.
some coniferous trees also showed an opposite respiratory
fractionation compared with leaf d13CR of deciduous trees. For
instance, d13CR of Pinus radiata needles (plus stem) was 13Cdepleted by 3.7& compared with OM (Troughton et al., 1974),
but this was not the case for other coniferous needles analyzed,
probably because the stem was included together with needles in the
case of P. radiata. Devaux et al. (2009) extracted high amounts of
pinitol from the phloem sap of Pinus pinaster. As pinitol is a
metabolite 13C-depleted compared to sugars, one could suggest
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
that its use for respiration explains the 13C depletion in CO2
respired by leaves and roots of conifers compared with other trees.
However, more data on conifers are needed and labelled pinitol
should also be used to test this hypothesis.
Intriguingly, during germination in darkness, potato tubers and
sprouts (which are heterotrophic organs, as are roots) also show a
13
C depletion in respired CO2 compared with starch (MaunouryDanger et al., 2009). For C4 plants, almost no fractionation in
maize roots was observed when they were cultured in nutrient
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New
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solution (Werth & Kuzyakov, 2005), while root-respired CO2 was
13
C-depleted up to 7& in different C4 species (including maize)
cultured in a mix of sand, perlite and soil (Schnyder & Lattanzi,
2005; Zhu & Cheng, 2011).
Similar to leaves, the nature of the organic material used as a
presumable source (OM vs WSOM or a carbohydrate fraction)
for the determination of apparent discrimination in roots does not
impact on these main findings (Fig. 2). In 10 instances of roots of
C3 herbaceous plants for which the isotopic signature of OM as
well as WSOM or a carbohydrate fraction was reported, only one
showed a difference in the sign of the apparent discrimination
between calculation on an OM basis and calculation on a
WSOM/carbohydrate basis. For these cases, OM was, on average,
0.71& more negative than WSOM or the carbohydrates. Thus,
apparent fractionation calculated on a WSOM or carbohydrate
basis qualitatively matches the results on an OM basis but leads to
a lower estimate of discrimination. An exception to this was
Arachis hypogea, with apparent discrimination on a WSOM basis
leading to depleted (1.08&) respiratory CO2 in line with all other
measurements, while on an OM basis a slight enrichment
( 0.52&) was observed. In 14 cases of roots of C3 woody species
for which the isotopic signature of OM as well as WSOM or a
carbohydrate fraction was reported, only one differed in the sign
of the apparent discrimination between calculation on an OM
basis and calculation on a WSOM/carbohydrate basis. OM was,
on average, 0.62& more negative than WSOM or the carbohydrates (see Fig. 2).
Based on these initial studies, the following patterns appear to
emerge: although both leaf and root respiratory apparent
fractionations are variable among species, they have opposite
signs (with few exceptions), that is, root respiratory fractionation
values are positive in C3 plants (13C depletion in root d13CR
compared with root OM), except for deciduous trees and shrubs,
while leaf respiratory fractionation is negative (13C enrichment in
leaf d13CR compared with leaf OM) except for some pine trees.
The difference in mean apparent respiratory fractionation
between leaves and roots is significant at P < 0.001 for C3 as
well as for C4 herbs (Wilcoxon test), while leaf and root apparent
respiratory fractionations are not significantly different for C3
woody species (P > 0.1). Given this difference, the question arises
as to when the leaf–root d13CR divergence appears during plant
ontogeny.
VI. d13CR of leaves and roots diverges at leaf
autotrophy onset
Interestingly, d13CR values of both leaves and roots were shown
(Fig. 5) to be close to the isotopic composition of seed OM
(c. 26&) in bean seedlings, but diverged upon leaf autotrophy
onset: leaf-respired CO2 became progressively 13C-enriched,
reaching values of c. 20& when leaves were fully expanded,
while that of roots became 13C-depleted, reaching values of
c. –29&, corresponding to the values already reported for adult
plants (Bathellier et al., 2008). Similar results were also obtained on
peanut seedlings, with leaves and roots having d13CR values similar
to those of bean plants despite more negative d13C values for peanut
New Phytologist (2014) 201: 751–769
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4
ΔR (‰) = δ13CS – δ13CR
762 Review
2
0
–2
–4
Bean leaves
Bean roots
No fractionation
Peanut leaves
Peanut roots
–6
–8
–10
4
6
8
10
12
14
16
Developmental stages (BBCH)
Fig. 5 Changes in apparent respiratory fractionation (DR) during early
ontogeny calculated as the isotopic difference between the putative
substrate (d13CS) and respired CO2 (d13CR) for bean (squares) and peanut
(circles) roots (closed symbols) and leaves (open symbols). The horizontal
solid line indicates no respiratory fractionation (i.e. DR = 0). Sucrose and
water-soluble fraction were taken as source carbon for respiration for bean
and peanut, respectively. During ontogeny, the d13C values of soluble
fraction varied from c. 27 to 29& and from c. 26 to 29.7& in peanut
leaves and roots, respectively. In bean plants, the d13C values of sucrose
changed from c. 25.5 to 27& and from c. 24 to 27& in leaves and
roots, respectively. As the growth rates were different between the two
species, the advance in phenological phases is expressed on the BBCH scale
(growth stage of mono-and dicotyledonous plants. Developmental stages;
BBCH monograph, 2001, Federal Biological Research Centre for Agriculture
and Forestry). The seedlings are heterotrophic until stage 10, which
corresponds to the beginning of leaf autotrophy. Values for bean plants are
from Bathellier et al. (2008) and for peanut from J. Ghashghaie et al.
(unpublished).
seeds (c. 29&) because of high lipid content (J. Ghashghaie et al.,
unpublished). By contrast, maize leaves show an opposite trend,
that is, 13C depletion in respired CO2 during ontogeny
(J. Ghashghaie et al., unpublished).
The observed divergence between root and leaf d13CR cannot
result from the difference in isotope composition of the respiratory
substrate between the two organs because sucrose becomes
13
C-depleted in both organs during leaf autotrophy acquisition;
that is, the isotopic difference between respired CO2 and sucrose
(putative substrate) increases in leaves, while it remains low and
constant in roots (Bathellier et al., 2008). Parallel changes in the
isotopic composition of bulk OM and sucrose in both leaves and
roots suggest that photosynthetic products transported from source
leaves to roots progressively change the isotopic signature of both
leaf and root OM but with a time lag as a result of the transport.
Changes in respiratory fractionation (DR) of roots and leaves
during ontogeny of peanut plants match quite well those observed
for bean plants (Fig. 5). Obviously, respiratory fractionation, being
negligible for both organs at the beginning of germination, changes
in opposite directions upon leaf autotrophy onset; that is, DR
becomes more negative in leaves, reaching 8& in both species
and positive in roots up to + 3.5&, presumably because of
differences in respiratory metabolic pathways between autotrophic
and heterotrophic tissues.
Ó 2013 The Authors
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dehydrogenase fractionates against 13C by c. 9.6& during
decarboxylation of C-1 of glucose (Rendina et al., 1984). Because
it originates from C-3 and C-4 positions of glucose, the C-1 of
pyruvate decarboxylated by PDH is expected to be 13C-enriched.
However, when pyruvate is only partly engaged in the PDH
reaction, the isotope effect of this enzyme will operate at its
maximum level, leading to a 13C depletion in CO2 evolved up to
23.8& compared with pyruvate (the in vitro value reported by
Melzer & Schmidt, 1987). Also, several enzymes associated with
the TCA cycle could fractionate against 13C (i.e. citrate synthase
fractionates by c. 20& (Tcherkez & Farquhar, 2005), NADPdependent isocitrate dehydrogenase fractionates by up to 5.7&
(Lin et al., 2008) and 2-oxoglutarate dehydrogenase may fractionate by c. 20& (Tcherkez & Farquhar, 2005)), so that TCA cyclederived CO2 is clearly 13C-depleted. In addition, the anaplerotic
CO2 fixation by PEPc may influence the isotope composition of
TCA cycle intermediates, unless all the PEPc-derived carbon atoms
within oxaloacetate molecules are subsequently decarboxylated
(Edwards et al., 1998). Taking all these effects together, rootrespired CO2 is expected to be generally 13C-depleted compared
with substrates. The question is how the changes in relative
activities of these decarboxylating pathways might affect overall
root d13CR.
Positional labelling experiments on attached roots of bean plants
immersed in glucose or pyruvate solutions 13C-labelled in C-1, C-2
or C-3 were conducted to estimate the relative contributions of
PDH, the TCA cycle and PPP to the overall root respiration (see
Fig. 6, redrawn from Bathellier et al., 2009). Labelling experiments
under typical dark conditions revealed an important PPP activity
(c. 22% of the overall respiration, the same rate as previously
reported for maize root tips (24%) by Dieuaide-Noubhani et al.,
1995). Similar experiments after a few days of continuous darkness
showed that the prolonged dark treatment mainly affected the TCA
cycle, which seemed to become notably reduced and fuelled mainly
by the lipid/protein recycling, and the ongoing synthesis of
VII. Metabolic pathways potentially implied in 13C
depletion in root-respired CO2
Recently, the metabolic origin of root d13CR compared with leaf
d13CR was investigated in bean plants (Bathellier et al., 2009).
Surprisingly, root d13CR does not follow the leaf d13CR pattern,
remaining low and almost stable whatever the carbohydrate pool
size during a prolonged dark period (Figs 3, 4c,f). Indeed, despite
a decrease in respiration rate (Fig. 4a) indicating a decrease in
carbohydrate pool size, and despite substantial changes in RQ
(Figs 3, 4b) suggesting a substrate switch from carbohydrates to
less oxygenated metabolites (e.g. lipids), root d13CR did not
change (Figs 3, 4c). Similar results were also observed when roots
were detached to prevent assimilate supply from leaves (Bathellier,
2008). This cannot originate from variation in the d13C value of
root metabolites, which might compensate for the switch of
respiratory substrate, because all the major root metabolites had
invariant d13C (see Fig. 1 in Bathellier et al., 2009). Clearly, leaves
and roots do behave differently, presumably because of differences in respiratory metabolic pathways between autotrophic and
heterotrophic tissues. Obviously, root respiration under starvation
involves metabolic changes that nevertheless result in respired
CO2 with d13C similar to that under typical nonstarving
conditions.
13
C depletion in root d13CR could be partly explained by
‘fragmentation fractionation’ during decarboxylation reactions
involved in respiratory pathways; that is, the TCA cycle releases
light carbon atom positions of glucose (C-1, C-2, C-5 and C-6) and
the pentose phosphate pathway (PPP) releases C-1 of glucose.
However, recent results on differences in isotopomer frequencies of
C-1 in glucose extracted from different organs (Gilbert et al., 2012)
point to the need for further studies on the variability of d13C
released from C-1 glucose during PPP activity. In addition, isotope
effects of the enzymes involved in decarboxylations could operate,
releasing 13C-depleted CO2. In the PPP, 6-phosphogluconate
PosiƟonal labelling of roots with:
13C-1
13C-2
glucose or
13C-3 pyruvate
Fig. 6 Positional labelling of root respiration
using glucose or pyruvate labelled at C-1, C-2
or C-3 positions (adapted from Bathellier
et al., 2009). Labelled carbon atom positions
are presented as grey circles and
corresponding labelled CO2 evolved as grey
ellipses. Biosynthetic pathways corresponding
to the synthesis of lipids and secondary
metabolites from acetyl-CoA, and
biosynthesis of amino acids (e.g. aspartate and
glutamate) from tricarboxylic acid (TCA) cycle
intermediates, as well as the pentose
phosphate pathway (PPP) are indicated by
dashed lines. The anaplerotic pathway for (re)
fixation of CO2 (after its hydration to HCO3 )
by phosphoenolpyruvate (PEPc) to feed the
TCA cycle with oxaloacetic acid (OAA) is also
presented.
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
13C-2
Glucose
Glucose
1 23456
1 23456
PPP
CO2
Pyruvate
CO2
PEP
CO3H-
1 23456
CO2
CO3H-
CO2
PEP
Malate
Acetyl-CoA
PEPc
Biosyntheses
Biosyntheses
CO2
Aspartate
Glutamate
Biosyntheses
OAA
Malate
CO2
CO2
PDH
Acetyl-CoA
TCA
cycle
CO2
32 1
CO3H-
CO2
PDH
PEPc
OAA
PPP
Pyruvate
CO2
32 1
Acetyl-CoA
PEPc
Aspartate
Glucose
PEP
PDH
glucose or
pyruvate
13C-1
PPP
Pyruvate
CO2
3 2 1
13C-3
glucose or
pyruvate
TCA
cycle
Aspartate
CO2
OAA
Malate
TCA
cycle
CO2
Glutamate
Glutamate
CO2
CO2
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764 Review
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the labelling experiments and calculations, see Bathellier et al.,
2009).
Based on the data discussed earlier, two pathways importantly
involved in root-respiratory processes could be at the origin of 13C
depletion in root-respired CO2 (i.e. PPP and the anaplerotic
pathway). The role of a higher PPP activity in roots (compared with
leaves) on the 13C depletion in overall root-respired CO2 has
already been studied (as discussed in the three preceeding
paragraphs). However, the potential impact of the anaplerotic
pathway on root d13CR is still unknown. Indeed, CO2 (re)fixation
by PEPc discriminates in favour of 13C (malate formed is thus 13Cenriched compared with other organic acids of the TCA cycle
coming from acetyl-CoA), leaving behind 13C-depleted CO2,
which should lead to 13C depletion in root net respired CO2. This
should also lead to 13C enrichment in root compared with leaf OM
(for a mass balance estimation, see Badeck et al., 2005). The impact
of CO2 fixation by PEPc will, however, depend on the rate of
reassimilation of respired CO2 by this enzyme.
glutamate was sustained by the anaplerotic action of PEPc, with no
effect on overall root d13CR (Bathellier et al., 2009). Fig. 7
summarises the relative activities of the metabolic pathways
involved in respiration and the prevalence of decarboxylations
leading to 13C enrichment or 13C depletion in overall respired CO2
from roots (left-hand panels) and leaves (right-hand panels) under
typical dark conditions (beginning of the night) or after a
prolonged period of darkness. Root d13CR was estimated to range
between c. 27& and c. 30& whatever the dark conditions,
because the main contributors to the respiration (PPP, TCA cycle
and lipid degradation) result in evolution of 13C-depleted CO2 as a
result of the ‘fragmentation fractionation’ and the isotope effects of
the enzymes involved. This is in agreement with the values observed
by Bathellier et al. (2009), shown on Figs 3 and 4(c). These authors
suggested that the observed invariance in the isotope composition
of root-respired CO2 under continuous darkness could be driven
by compensations between both the different fractionating steps
and the composition of the respiratory substrate mix (for details of
ROOTS
LEAVES
Beginning of the dark period
Glucose
PPP
CO2
PEP
CO2
ME
HCO3
-
PDH
ME
CO2
Aspartate
Biosyntheses
OAA
TCA
cycle
Malate
PDH
CO2
Acetyl-CoA
PEPc
Pyruvate
HCO3-
CO2
CO2
Acetyl-CoA
PEPc
Biosyntheses
Aspartate
CO2
CO2
PEP
CO2
Pyruvate
PPP
Glucose
OAA
TCA
cycle
Malate
CO2
Glutamate
Glutamate
CO2
CO2
After prolonged dark period
CO2
PEP
CO2
ME
ME
Pyruvate
HCO3
CO2
PDH
Aspartate
CO2
Acetyl-CoA
Lipids
PEPc
Malate
PDH
CO2
Acetyl-CoA
PEPc
OAA
TCA
cycle
Pyruvate
HCO3-
CO2
CO2
PEP
CO2
-
PPP
Glucose
PPP
Glucose
Aspartate
CO2
Lipids
OAA
Malate
TCA
cycle
CO2
Glutamate
CO2
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Glutamate
CO2
Fig. 7 Metabolic pathways involved in root
(left panels) and leaf (right panels) respiration.
The relative importance of the pathways
reported by Bathellier et al. (2009) for roots
and by Tcherkez et al. (2003) for leaves of
Phaseolus vulgaris under typical dark
conditions (upper panels) and after prolonged
darkness (lower panels) is shown by the
thickness of the arrows. The grey areas
indicate the prevalence of decarboxylating
pathways for each case, with pyruvate
dehydrogenase (PDH) and malic enzyme (ME)
releasing 13C-enriched CO2 while the
tricarboxylic acid cycle (TCA) and the pentose
phosphate pathway (PPP) release 13Cdepleted CO2. Degradation of lipids feeds the
TCA cycle with 13C-depleted molecules as
well. In the case of leaf respiration under
typical conditions, decarboxylation of malate
by ME is high at the light-to-dark transition but
decreases rapidly in the dark. Biosynthesis of
amino acids (e.g. aspartate and glutamate)
from TCA cycle intermediates and the
resulting anaplerotic feeding of the TCA cycle
with oxaloacetic acid (OAA) via
phosphoenolpyruvate carboxylase (PEPc)
activity are also shown.
Ó 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
New
Phytologist
Furthermore, respiratory metabolism is affected by nitrogen
nutrition (NO3 or NH4+) through acid–base regulation, cellular
pH stat and the related activities of carboxylases as well as the fate of
carbon introduced by these carboxylases (Raven & Farquhar,
1990). Cramer et al. (1993) demonstrated that incorporation of
14
C in maize roots is higher when plants are cultured with NH4+. To
maintain the cellular pH and supply carbon for biosynthesis of
amino and organic acids, CO2 fixation by PEPc via the anaplerotic
pathway is activated. Schweizer & Erismann (1985) observed
opposite effects of N nutrition on PEPc activity in leaves vs roots of
nonnodulated bean plants; PEPc activity was low under NH4+ and
high under NO3 nutrition in primary leaves but the reverese was
seen in roots. With a proteomic study on starved maize plants,
which were subsequently subjected to high NO3 concentration in
the culture medium, Prinsi et al. (2009) suggested that the
nutritional status of the plant may affect two different posttranslational modifications of PEPc, consisting of monoubiquitination (thus reduction of its affinity for PEP) and phosphorylation
(activation) in roots and leaves, respectively. They also showed
increased amounts of the enzymes involved in PPP (i.e. glucose
6-phosphate dehydrogenase and 6-gluconate dehydrogenase) in
roots and proteins involved in the regulation of photosynthesis and
also lipid metabolism in leaves. Further investigations on the
impact of the anaplerotic pathway on the isotopic signature of both
root OM and root-respired CO2 under varying nitrogen nutrition
conditions are needed to understand its potential contributions to
the between-organ isotopic differences in different species.
In addition, as mentioned earlier, C4 leaves discriminate only
slightly during respiration (in contrast to C3 leaves). It would be
interesting to examine whether this is related to the high PEPc
activity in C4 leaves (analogous to roots having higher PEPc activity
and lower respiratory fractionation).
The measurement of root respiration, and specifically of the
isotopic signature of root-respired CO2, raises a series of methodological issues because it requires separation of the root-derived
signal from that resulting from soil microbial activity. Two main
approaches to tackle this issue have been employed so far in the
studies cited here. Some researchers used quasi-sterile growth
media (sterilized sand or nutrient solutions) while others extracted
the roots from the soil and cleaned them before measuring
respiration (see Table 1). The advantage of the first method is that
in situ measurements on the soil/root system can be done, avoiding
potential artefacts as a result of extraction of roots and cleaning.
However, this comes at the price of a restricted range of soil types
that can be studied. When applying the second method, it cannot
be ruled out that the separation of the roots from their in situ
environment can lead to alterations in root respiratory metabolism.
In particular, if the commitment of different catabolic and
anaplerotic pathways depends on ion exchange with the soil
medium, exposing the roots to air can be expected to lead to
adjustments in the respiratory metabolism. In addition, if roots fix
CO2 through PEPc, the fraction of assimilated carbon stemming
from respired organic material and thus carrying relatively negative
signatures relative to the fraction stemming from ambient air that is
less depleted in 13C is most likely going to change after extraction of
roots from the soil. In consequence, the isotopic signature of rootÓ 2013 The Authors
New Phytologist Ó 2013 New Phytologist Trust
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Review 765
respired CO2 will change. Currently little is known about these
potential artefacts and the timescales on which they establish.
VIII. A glance at stem respiration
Plant stems connect leaves and roots, providing transport of water
and minerals from roots to leaves, transport of assimilates from
leaves to roots, and mechanical stability to the above-ground plant
organs anchored within the soil. Very often plant stems also contain
photosynthetically active tissues and can store reserves. As such,
stems are characterized by a complex mix of vertical and radial gas
transport pathways as well as often relatively high distances of living
cells from the stem surface. In consequence, CO2 exchange fluxes
across the stem surface are determined by the stem internal CO2
partial pressure gradients, which, in turn, depend on respiration of
diverse stem tissues, CO2 exchange with the fluids transported
within xylem and phloem, and stem photosynthesis. In a similar
manner, the putative substrates of stem respiration also vary with
local stem photosynthesis, import from phloem sap and export to
phloem sap. An in-depth discussion of the current state of
knowledge about fractionation during stem respiration goes
beyond the scope of current review. Here, we only report empirical
results on apparent fractionation without addressing the issue of
identification of the source carbon. Some aspects of the mentioned
issues related to CO2 exchange fluxes across the stem surfaces are
dealt with in recent reviews and papers by Teskey et al. (2008) on
transport of CO2 within tree trunks, by Cernusak et al. (2001) on
stem photosynthesis, and by Damesin et al. (2005) on different
methods for CO2 sampling.
Stem-respired CO2 of C3 species is, in most cases, 13C-enriched
(Fig. 1c, see Notes S2 for references used). Thus, apparent
respiratory discrimination in stems resembles the phenomenon
described for leaves with a lower average DR of 1.65&, as
compared with a mean of 3.8& in leaves. The set of measurements currently available is dominated by experiments on tree
stems of mainly European tree species.
Leaf and stem respiration during ontogeny of current-year
shoots of Fagus sylvatica from sleeping buds to 3 months after budburst showed negative DR for both organs throughout the
observation period (Eglin et al., 2009). Heterotrophic buds had
higher d13CR because 13C-enriched reserves were used, while
respired CO2 of photosynthesizing leaves and stems progressively
became more 13C-depleted after bud-burst.
IX. Impact on carbon isotope composition of plant OM
Post-photosynthetic discrimination, discussed earlier, could lead to
13
C depletion in the bulk OM of autotrophic organs (i.e. leaves)
relative to the average isotopic composition of photosynthetic
assimilates, mainly because of generally 13C-enriched CO2 evolved
during the night. An opposite trend will result from exhalation of
13
C-depleted volatile compounds evolved from leaves or ablation
of leaf waxes in some species. Heterotrophic organs could become
13
C-enriched mainly because of 13C-depleted CO2 evolved via PPP
during respiration, CO2 (re)fixation by PEPc (Badeck et al., 2005)
and probably because of the transport of 13C-enriched assimilates
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from autotrophic source to sink heterotrophic organs (Gessler
et al., 2007). Such changes in the isotopic signature of organic
material, as a result of post-photosynthetic discrimination
compared with that of photosynthetic primary products carrying
the isotopic imprint of photosynthetic discrimination, could
introduce bias in the use of such signatures as references in both
ecosystem partitioning studies and in the estimation of water-use
efficiency.
However, the impact of post-photosynthetic discrimination on
the isotopic signature of OM has not been experimentally
demonstrated as yet, except by Nalborczyk (1978), who experimentally showed for different plant species that the 13C : 12C ratio
(natural abundance) in leaf OM increases with increasing rates of
14
C-labelled CO2 fixation by PEPc in the dark (Fig. 8). Further
investigations on the potential impact of discrimination during
different post-photosynthetic processes on isotope composition of
plant OM are needed to improve our interpretation of the isotopic
signature of plant OM as well as between-organ differences.
X. Conclusions
Despite high variability in both leaf d13CR and root d13CR, and
despite few exceptions for both organs, the present review has
collected evidence of opposite ‘apparent’ carbon isotope discrimination during respiration in roots as compared with leaves in
herbaceous species. In contrast to leaves, which in general release
13
C-enriched respired CO2 compared with leaf OM, the CO2
evolved by roots is 13C-depleted compared with root material. This
could partly explain the between-organ isotopic difference already
reported in the literature.
Interestingly, significant differences between functional groups
(C3 herbs vs C3 woody species, and C3 vs C4 herbs) are seen mainly
for roots. While leaf d13CR is 13C-enriched in both C3 herbs and C3
woody species (and also in some C4 herbs), roots of C3 herbs show
–20
δ13C of leaves (‰)
–21
–22
opposite respiratory fractionation compared with roots of C3
woody plants – root d13CR being 13C-depleted in C3 herbs and
13
C-enriched in C3 woody species compared with root material.
The respiration of mycorrhizas associated with tree roots could
explain the opposite respiratory fractionation observed between
roots of C3 herbs and C3 woody species.
The route to further progress in exploring the causes of these
patterns will involve scrutinising potential artefacts during measurements of root respiration; assessing the role of PEPc activity;
and studying differences in root fractionation processes in soils/
substrates differing in pH and ammonium vs nitrate availability.
The divergence in 13C between leaves and roots in C3 herbs is
shown to establish upon the heterotrophy–autotrophy transition,
suggesting that heterotrophic tissues/organs behave differently
from autotrophic ones with respect to respiratory metabolism. The
relative activities of metabolic pathways releasing 13C-enriched or
13
C-depleted CO2 (PDH and malic enzyme vs TCA cycle and
PPP) are shown to be at the origin of the isotopic signature of leaf- vs
root-respired CO2. Metabolic studies similar to those discussed
should be conducted during ontogeny to elucidate the metabolic
origin of the observed divergence at autotrophy onset.
The analyses of apparent respiratory discrimination based on
OM as reference material or, alternatively, based on carbohydrates
or WSOM as putative substrate for experiments that provided both
the isotopic signature of OM and carbohydrates or WSOM did not
provide any evidence of contradictory results on apparent respiratory discrimination between the use of the two groups of
references. The sign of the apparent respiratory discrimination
essentially did not change, while the absolute magnitudes differed
as a result of systematic differences between the isotopic signatures
of OM and carbohydrates.
The results presented in the current paper help to constrain the
analysis of carbon isotope exchange fluxes in ecosystems with
multiple sources of respiratory CO2 (Ogee et al., 2003; Werner
et al., 2007; Wingate et al., 2010) and allow a better understanding
of the origin of soil CO2 isotopic signatures (Br€
uggemann et al.,
2011). Furthermore, they indicate that there are good prospects for
the development of on-line carbon isotope exchange measurements
(Barbour et al., 2007), which can be applied to in vivo diagnosis of
active metabolic pathways.
–23
Acknowledgements
–24
The authors are grateful for financial support through the SIBAE
network (COST Action: COST ES0806) coordinated by Nina
Buchmann, which facilitated fruitful discussions with the SIBAE
members. We also thank these colleagues for their encouragement
to prepare a review on this topic. Many thanks also to the colleagues
who kindly shared their data (even unpublished in some cases).
–25
y = 3.66x –27.06
R2 = 0.9162
–26
–27
0
2
4
6
Dark carboxylation (nmol
13
8
14CO
2
10
g–1
DW
12
s–1)
Fig. 8 Carbon isotope composition (d C) of leaf bulk organic matter as a
function of carboxylation rate in the dark, measured using 14C-labelled CO2
on sunflower, tomato, rape, barley, rye, cucumber, wheat and lupine. d13C
was determined in ambient air before labelling on plants grown under low
light (drawn using data from Nalborczyk, 1978).
New Phytologist (2014) 201: 751–769
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Supporting Information
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version of this article.
Notes S1 Leaf isotopic data used for Fig. 1(a) are from the
references listed in supporting information.
Notes S2 Stem isotopic data used for Fig. 1(c) are from the
references listed in supporting information.
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