Phytochemistry Reviews 2: 145–161, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
145
Carbon isotope fractionation during dark respiration and
photorespiration in C3 plants
Jaleh Ghashghaie1,∗ , Franz-W. Badeck2 , Gary Lanigan3 , Salvador Nogués1 , Guillaume
Tcherkez1 , Eliane Deléens4,† , Gabriel Cornic1 & Howard Griffiths3
1 Département
d’Ecophysiologie Végétale, ESE, CNRS-UMR 8079, Bât.362, Université de Paris XI, 91405Orsay Cedex, France; 2 Potsdam Institute for Climate Impact Research (PIK), P.O. Box 601203, 14412 Potsdam,
Germany; 3 Department of Plant Sciences, Downing Street, University of Cambridge, Cambridge CB2 3EA,
United Kingdom; 4 Laboratoire de Structure et Métabolisme des Plantes, IBP, Université de Paris XI, 91405Orsay Cedex, France; ∗ Author for correspondence (Tel: ++ 33 1 69 15 63 59; Fax: ++ 33 1 69 15 72 38; E-mail:
[email protected])
Key words: carbon isotope, C3 plants, discrimination, fractionation, isotope effect, photorespiration, respiration
Abstract
Carbon isotope discrimination during photosynthetic CO2 assimilation has been extensively studied and rigorous
models have been developed, while the fractionations during photorespiratory and dark respiratory processes have
been less well investigated. Whilst models of discrimination have included specific factors for fractionation during
respiration (e) and photorespiration (f ), these effects have been considered to be very small, i.e. not significantly
modifying the net discrimination expressed in organic material. On this paper we consider the fractionation effects
associated with specific reactions set against the overall discrimination which occurs during source-product transformations. We review the studies which have recently shown that discrimination occurs during respiration at night
in intact C3 leaves, leading to the production of CO2 enriched in 13 C (i.e., e = −6), and modifying the signature
of the remaining plant material. Under photorespiratory conditions (i.e. increased oxygen concentration and high
temperature), the photorespiratory fractionation factor may be high (with f around +10), and significantly
alters the observed net photosynthetic discrimination measured during gas exchange. Fractionation factors for both
respiration and photorespiration have been shown to be variable among species and with environmental conditions,
and we suggest that the term ‘apparent fractionation’ be used to describe the net effect for each process. In this
paper we review the fractionations during photorespiration and dark respiration and the metabolic origin of the
CO2 released during these processes, and we discuss the ecological implications of such fractionations.
Introduction
Carbon has two stable (non-radioactive) isotopes; the
predominant one is 12 C (natural abundance is about
98.9%), and the minor one is 13 C (about 1.1%). During photosynthetic CO2 assimilation, a number of
isotope effects (see Appendix for definitions) discriminate against the heavier stable isotope, leading to
† This paper is dedicated to Eliane Deléens who sadly passed
away in March 2003. She initiated work on discrimination during
dark respiration at the University of Paris-XI (Orsay-France). She
was a pioneer in the use of stable isotopes in plant ecophysiological
studies.
photosynthetic products being depleted in 13 C compared to atmospheric CO2 . The carbon isotope composition (δ 13 C) of plant material varies from −7 to
−35, largely depending on photosynthetic pathway
(C3, C4 or CAM), with considerable variation in δ 13 C
within each of these plant groups. This variation in
C3 species is primarily dependent on plant species,
anatomical characteristics and environmental conditions (Farquhar et al., 1989; Brugnoli and Farquhar,
2000). Further fractionations also occur during anabolic and catabolic metabolism, leading to different
isotopic signatures for biochemical compounds. Thus,
146
for example, lipids are 13 C-depleted compared to carbohydrates (Abelson and Hoering, 1961; Park and
Epstein, 1961; Deléens et al., 1984; Gleixner et al.,
1993). Variations also occur within a single plant, such
that leaves are generally 13 C-depleted compared to all
other organs. This difference between organs could
be attributed to a possible fractionation during export
of assimilates (phloem loading) and/or to a possible
fractionation during dark respiration, which may vary
between organs.
Carbon isotope discrimination () is used to define
the net shift in carbon isotope composition during photosynthetic CO2 assimilation in C3 plants, from source
CO2 to organic material, and has been extensively investigated and defined by mathematical models (see
Appendix, also Vogel, 1980; O’Leary, 1981; Farquhar et al., 1982, 1989). This overall discrimination
involves fractionation during both physical and biochemical processes. The complete theory for the net
photosynthetic discrimination is described as follows
(Farquhar et al., 1982):
= ab
b
Pa − Ps
Ps − Pi
Pi − Pc
+a
+ (es + ai )
+
Pa
Pa
Pa
Pc
−
Pa
eRd
k
+ f ∗
,
Pa
(1)
where, Pa , Ps , Pi and Pc are the CO2 partial pressures
in the ambient air, at the leaf surface, in the intercellular air spaces (gas phase) and in the chloroplasts at the
carboxylation sites (liquid phase), respectively. Accordingly, ab is the fractionation during CO2 diffusion
in the boundary layer (= 2.9, Farquhar, 1980); a is
the fractionation during CO2 diffusion in air through
stomata into the leaf (= 4.4, Craig, 1954); es is
the fractionation occurring when CO2 is dissolved in
the cell solution (= 1.1 at 25 ◦ C, Vogel, 1980); ai
is the fractionation during CO2 diffusion in the liquid
phase (0.7, O’Leary, 1981); b is the net discrimination by the primary carboxylating enzymes ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco)
and phosphoenolpyruvate carboxylase (PEPc) during
carboxylation in C3 plants (b varies between 28.2
and 30, assuming that carboxylation by PEPc varies respectively between 5% and 0%, Brugnoli et al.
1988); e and f denote overall discriminations during day respiration (Rd ) and photorespiration, relative to photosynthetic products, respectively; k is the
carboxylation efficiency and ∗ is the CO2 compensation point in the absence of day respiration (Brooks
and Farquhar, 1985).
There is uncertainty regarding the magnitude of
respiratory and photorespiratory fractionations (terms
‘e’ and ‘f ’, respectively) and, because of difficulties
in experimentally determining these values, a simple
model is usually used to approximate the entire theory. The simplified model (Farquhar et al., 1982) only
includes two main discriminating steps; CO2 diffusion from the air, via stomata, into the leaf air spaces
and net carboxylation, and allows us to predict instantaneous discrimination (i ) from measurements
of Pi /Pa :
i = a + (b − a)
Pi
Pa
(2)
According to this model, all factors decreasing stomatal conductance and consequently decreasing Pi /Pa
should also linearly decrease photosynthetic discrimination. The simple model is remarkably robust under
a range of experimental conditions (see review of
Brugnoli and Farquhar, 2000 and references therein),
demonstrating a high correlation between Pi /Pa and
δ 13 C of the photosynthetic products under varying
light intensities, vapour pressure deficit or under water
deficit conditions. When the values for ‘b’, determined
by in vitro measurements are used, the net photosynthetic discrimination measured on-line in an open gas
exchange system often deviates from the values predicted by Eq. 2 (Evans et al., 1986; von Caemmerer
and Evans, 1991). This deviation results from (i) a
mesophyll conductance component, leading to an additional drawdown in CO2 partial pressure from the
substomatal cavity to Rubisco (Pi to Pc ); (ii) uncertainties in the estimation of Pi /Pa because of heterogeneous stomatal closure and/or a substantial cuticular
conductance to water vapour under water deficit conditions (for a recent review see Evans and Loreto,
2000) and (iii) the effect of fractionation which may
occur during photorespiratory and respiratory processes on the net photosynthetic discrimination, and
extent of refixation of respiratory CO2 .
During real-time measurements, Evans et al.
(1986) showed that the difference between the observed net photosynthetic discrimination measured
with an on-line system and the values predicted by
the simple model was a linear function of A/Pi (A
is the net photosynthetic CO2 assimilation), the slope
being related to the mesophyll conductance and the
intercept to the (photo)respiratory fractionation (the
last term in Eq. 1). Their method is now routinely
used for the estimation of leaf mesophyll conductance.
Since the intercept is usually small, it is considered
147
that the fractionation during photorespiration (term
‘f ’) and day respiration (term ‘e’) is negligible and
does not significantly modify the net photosynthetic
discrimination measured on-line, particularly since
measurements are usually made under 1–2% O2 to
inhibit photorespiration. However, we discuss the implications of photorespiration and day-time respiration
and their associated fractionations below.
The carbon isotope signature of plant dry matter
integrates not only the discrimination during net CO2
assimilation in the light but also the discrimination that
could occur during the night-time respiration. Therefore, any fractionation during the night and/or the use
of heavy or light substrates for dark respiration (releasing 13 C-enriched or 13 C-depleted CO2 compared
to leaf material) should change the isotopic signature of the remaining leaf material. Moreover, when
non-photosynthesising organs are taken into consideration, release of 13 C-enriched or 13 C-depleted CO2
will further contribute to changes in the carbon isotopic signature of the whole plant. Henderson et al.
(1992) observed on some C4 species that the discrimination determined on leaf dry matter was significantly
greater than that measured on-line. Using a modelling
approach, they proposed that at least a part of this difference could be explained by the fractionation during
dark respiration releasing CO2 enriched in 13 C relative to the plant material. Any possible fractionation
during export could also contribute to this difference,
but to our knowledge, this has not been experimentally
demonstrated.
The potential changes of isotopic signatures in
plant organic matter due to dark respiratory processes
are of relevance for the use of 13 C in ecosystem studies. The models applied for these studies often build
on the assumption that the signature of organic matter is determined by the signature of photosynthetic
products, i.e. respiratory metabolism during the night
does not alter the signature of organic matter left behind. Although Lin and Ehleringer (1997) found no
apparent fractionation during dark respiration in mesophyll protoplasts (isolated from mature leaves of C3
and C4 plants) incubated with substrates of known
δ 13 C (fructose, glucose or sucrose), Duranceau et al.
(1999) Ghashghaie et al. (2001) and Tcherkez et al.
(2003) recently showed a substantial 13 C-enrichment
in dark respired CO2 compared to leaf plant material
even compared to leaf sucrose, the most 13 C-enriched
respiratory substrate, on intact leaves of some C3
plants. Sucrose being the potential substrate for dark
respiration, they concluded that an apparent fraction-
ation during dark respiration occurs in the species
studied.
The discrimination during photosynthetic CO2 assimilation has already been recently reviewed (see
Brugnoli and Farquhar 2000). In this paper, we focus
on the discrimination which occurs during dark respiration and (photo)respiration (i.e., photorespiration and
day respiration) in C3 plants.
Fractionation during dark respiration
What is the ‘fractionation factor’ during dark
respiration?
In the broadest sense, the discrimination which occurs
during respiration can be determined from the release
of respiratory CO2 , as compared to the isotopic signature of the reference (source) carbon pool, which we
define as an ‘apparent fractionation’ (see Appendix).
Individual fractionation factors will be associated with
kinetic isotope effects, non-homogeneous isotope distribution in respiratory substrates (positional effects)
or oxidation of different substrate pools (for definitions, see Appendix). The respiratory process will also
lead to a change in the isotopic signature of the pool
left behind. Depending on the question that is being
addressed, the reference pool can be the total organic
matter, recent photosynthetic products or the substrates actually used for respiration. Because there is
currently no consensus defining the reference pool for
respiratory processes, in the following, we explicitly
define the reference pools we address. In particular,
the fractionation factor used in Eq. 1 for respiration (e)
relates to the offset in isotopic signal from the primary
carboxylase (Rubisco) product during day respiration.
While we use the term ‘e’ to indicate fractionation
factors associated with both day and dark respiration,
it should be noted that values of e quoted herein are
for apparent fractionation associated with dark respiration, while the values associated with day respiration
remain unknown. We now consider the magnitude of
e and whether in practise it should be defined as an
apparent fractionation.
Carbon isotope signature of CO2 respired in the dark
Only a few published studies have investigated the carbon isotope signature of CO2 respired in the dark by
plants, and most of them were conducted on seedlings
(see Table 1). To our knowledge, the earliest work
was done by Baertschi (1953) who showed that CO2
148
Table 1. Carbon isotope signature of CO2 respired in the dark in different plant species compared to that of plant material. 13 C-enrichment
(−) and 13 C-depletion (+) in respired CO2 compared to plant material is indicated by negative and positive signs, respectively, in the column
‘fractionation’.
Respired CO2 Plant material Plant part
δ 13 CR
δ 13 CP
analysed
()
()
C3 species:
Phaseolus sp. (bean)
Lycopersicon esculentum (tomato)
Curcubita moschata L. (Squash)
Arachis hypogaea L. (peanut)
−24.3
−25.7
−23.8
−33.3
Helianthus annuus L. (sunflower)
−30.2
−35.9
−26.0
Triticum aestivum L. (wheat)
−25.4
−24.0
−25.5
Ricinus communis L. (Castor bean) −27.5
−28.4
Pisum sativum L. (pea)
−25.8
−27.8
Raphanus sativus L. (radish)
−30.1
Nicotiana tabacum (tobacco)
−24.1
Pinus radiata
−25.7
Phaseolus vulgaris L. (French bean) −20.2
Nicotiana sylvestris
−23.5
Fagus sylvatica L. (beech)
−22.1
−26.3
−22.1
−26.3
C4 species:
Zea mays L.
−13.5
−12.9
−12.0
−15.7
Paspalum dilatatum
−14.9
Gomphrena globosa
−17.5
Fractionation Reference
δ 13 CP -δ 13 CR
()
−32.4
−26.7
−28.5
−27.9
−27.3
−25.6
−28.5
−23.7
−23.5
−30.4
−28.7
−28.7
−26.1
−25.9
−28.8
−25.5
−29.4
−26.0
−26.5
−24.8
−26.2
−22.8
−23.3
Germinating seeds
0
Whole plant
−8.1
8-day seedling
−1.0
Soaked seed
−4.7
32-day seedling
+5.4
Soaked seed
+2.9
10-day seedling
+10.3
Leaf sucrose pool
−2.5
Soaked seed
+1.7
7-day seedling
+0.5
Leaf + ears
−4.9
Soaked seed
−1.2
12-day seedling
−0.3
Soaked seed
−0.3
24-day seedling
+1.9
4-day seedling
+1.3
Leaf starch
−1.1
Needles + stem
+3.7
Leaf sucrose pool
−5.8
Leaf sucrose pool
−3.0
Young twigs (May)
−2.7
Young twigs (December) +0.1
Twig sucrose (May)
−0.7
Twig sucrose (December) +3.0
−12.2
−11.6
−13.4
−13.9
−14.7
−15.7
Soaked seed
13-day seedling
Leaf sucrose
Leaf
Leaf
Leaf
respired by germinating bean had the same 13 C/12 C
ratio as the whole seed. Later, Park and Epstein (1961)
observed that CO2 respired by tomato plants was 13 Cenriched compared to whole plant material by about
8. They also observed, as did Abelson and Hoering (1961), that lipids were 13 C-depleted compared to
other metabolites by about 7–8. In agreement with
conservation laws, they emphasised that any depletion in 13 C in one chemical component of the plant,
such as lipid, must be compensated by enrichment in
13 C
+1.3
+1.3
−1.4
+1.8
−0.2
+1.8
Baertschi (1953)
Park and Epstein (1961)
Smith (1971)
Smith (1971)
Smith (1971)
Smith (1971)
Smith (1971)
Ghashghaie et al. (2001)
Smith (1971)
Smith (1971)
Troughton et al. (1974)
Smith (1971)
Smith (1971)
Smith (1971)
Smith (1971)
Smith (1971)
Hsu and Smith (1972)
Troughton et al. (1974)
Duranceau et al. (1999)
Ghashghaie et al. (2001)
Damesin and Lelarge (2003)
Damesin and Lelarge (2003)
Damesin and Lelarge (2003)
Damesin and Lelarge (2003)
Smith (1971)
Smith (1971)
Ghashghaie (unpublished)
Troughton et al. (1974)
Troughton et al. (1974)
Troughton et al. (1974)
in some other component. Since respiration and
lipid formation are closely related biochemical systems, the 13 C-enrichment in respired CO2 could result
from lipid formation. They also suggested, on the basis
of their biochemical theory, that in the case of seeds
germinating in the dark reported by Baertschi (1953),
lipids are not being formed, but are being oxidised.
Therefore, the isotopic signature of CO2 respired is
expected to be that of seeds (sugars and lipids as a
whole). However, if 13 C-depleted lipid is being syn-
149
thesised rather than oxidised, the respired CO2 would
be expected to have a higher 13 C content than the
whole plant.
Similar experiments were conducted later (Smith,
1971; Hsu and Smith, 1972; Troughton et al., 1974)
on different species, for which the CO2 respired in
the dark was shown to be 13 C-enriched (1–8) or
13 C-depleted (1–10) compared to the source organic
material (seed, seedling, leaf, whole plant material or
carbohydrates) and varied between species (Table 1).
In a review, O’Leary (1981) suggested that at least
some of these variations may be due to the source
of CO2 used for respiration which might have a carbon isotope composition different from that of whole
plant material. One could also suggest that the enrichment or depletion of respired CO2 in 13 C compared to
leaf dry matter or leaf carbohydrates may be due to a
fractionation during dark respiration.
Huge changes in respired CO2 signature were also
reported in the above studies during seedling growth,
ageing and with dark period duration. Moreover, in
sliced potato tubers, the signature of respired CO2 varied with time (Jacobson et al., 1970). The observed
changes do presumably reflect a switch in the substrate
used for respiration and/or a possible change in the
respiratory fractionation.
Surprisingly, investigations on the isotopic signature of dark respired CO2 were halted for more than
20 years until Lin and Ehleringer (1997) showed that
no apparent fractionation occurred during dark respiration measured in vitro on isolated protoplasts. This
result reinforced the initial assumption made in the
simple discrimination model, i.e. net fractionation
during respiration (term ‘e’ in model, see Eq. 1) is
negligible.
However, Duranceau et al. (1999) recently observed in both control and dehydrated Phaseolus
vulgaris plants (Figure 1A) that the respired CO2
was consistently 13 C-enriched compared with the leaf
sucrose pool by about 6, whatever the leaf age and
the leaf relative water content. They suggested that, if
sucrose (or a closely linked metabolite) is used as the
main substrate for dark respiration, a constant apparent fractionation of about −6 occurs in these plants.
Ghashghaie et al. (2001) then showed that this varied
between species and with drought. Respired CO2 was
13 C-enriched compared to leaf sucrose in average by
about 3 in well-watered Nicotiana sylvestris and by
2–6 in control Helianthus annuus (Figure 1B and C,
closed symbols). Using an on-line gas exchange system (in normal air), Duranceau et al. (2001) observed
Figure 1. Relationship between carbon isotope composition (δ 13 C)
of CO2 respired in the dark and of leaf sucrose for well-watered
(full symbols) and dehydrated (open symbols) P. vulgaris (A)
N. sylvestris (B) and H. annuus (C) plants. Dashed line corresponds
to 1:1 relationship. Data on A are redrawn from Duranceau et al.
(1999) and those on B and C from Ghashghaie et al. (2001).
an apparent fractionation value in N. sylvestris which
was nearly identical to the values obtained by Ghashghaie et al. (2001) on the same species using a CO2 -free
closed system. This indicates that the use of a CO2 free system does not affect the signature of the dark
respired CO2 . Contrary to what had been observed
in P. vulgaris, the apparent respiratory fractionation
increased in dehydrated N. sylvestris and decreased
in dehydrated H. annuus compared to control plants
(Figure 1, open symbols). Ghashghaie et al. (2001)
concluded that (i) carbon isotope fractionation during
dark respiratory process is a widespread phenomenon
occurring in C3 plants, (ii) this apparent fractionation is not constant and varies among species and
also varies with drought, but differently among species
150
(constant in P. vulgaris, increased in N. sylvestris and
decreased in H. annuus under drought compared to
control conditions). Such a variable apparent fractionation observed during dark respiration is consistent
with data in the older literature (see Table 1).
Possible causes of the observed overall fractionation
during dark respiration
Apparent fractionation may be expected during dark
respiration because of (i) positional effects such as
non-uniform 13 C-distribution within the hexose molecules (Rossmann et al., 1991; Gleixner and Schmidt,
1997) and (ii) isotope effects during the pyruvate dehydrogenase (PDH) reaction (De Niro and Epstein,
1977; Jordan et al., 1978; Melzer and Schmidt, 1987).
Effect (i) can lead to 13 C-enriched respiratory CO2 ,
effect (ii) can result in 13 C-depleted respiratory CO2 .
The overall effect will depend, as we are going to
discuss below, on the relative activities of different
metabolic pathways.
(i) Non-uniform 13 C-distribution within hexose
molecules
Abelson and Hoering (1961) initially suggested that
there was a homogeneous 13 C-distribution in hexose
molecules. Rossmann et al. (1991) experimentally
demonstrated that the C-3 and C-4 of glucose molecules (commercial glucose extracted from sugar beet
syrup and hydrolysed from maize flour) were enriched
in 13 C compared to other carbon positions which they
related to an isotope effect during aldolase reactions.
In the course of lipid biosynthesis, the pyruvate issued
from glucose by the glycolytic pathway produces 13 Cenriched CO2 by decarboxylation of the 13 C-enriched
carbons (C-3 and C-4 coming from glucose). The
more depleted sites (i.e., C-1, C-2, C-5 and C-6)
form acetyl-CoA which is subsequently oxidised in
the Krebs cycle or diverted to biosynthesis of other
metabolites (e.g., lipids) (Rossmann et al., 1991).
De Niro and Epstein (1977) have already emphasised
that, assuming such a heterogeneous 13 C-distribution
in glucose molecules, the well-known 13 C-depletion in
lipids compared to carbohydrates reported by Abelson
and Hoering (1961) and Park and Epstein (1961) thus
the 13 C-enrichment in CO2 produced during pyruvate
decarboxylation could be explained without recourse
to isotope effects during lipid metabolism. By analogy,
a constant shift of 1.3 to 1.7 between δ 13 C of ethanol in wine and sugars relative to the ‘must’ has been
observed in grape-wine. This fractionation has also
been attributed to decarboxylation of the 13 C-enriched
C3 and C4 positions during fermentation (Rossmann
et al., 1996).
In agreement with Park and Epstein (1961), we
suggest that the magnitude of the expected effect will
depend on the relative importance of the metabolic
pathways in plants. If a high fraction of catabolised
carbon is used for lipid biosynthesis (or other substances issued from acetyl-CoA), this should lead to
a high enrichment in 13 C of released CO2, conversely
if catabolised carbon is completely respired, all ‘light’
as well as ‘heavy’ carbon atoms of sugars will be decarboxylated. This pattern may explain the variability
in the values of δ 13 C of respired CO2 reported in the
literature (Table 1). This may also explain the results
of Lin and Ehleringer (1997): if, for isolated protoplasts, all the carbon atoms (heavy and light) from
sugars are consumed in the Krebs Cycle and no carbon
is directed towards the biosynthesis of metabolites,
such as lipids, the signature of overall dark respired
CO2 would then be the same as the signature of the
sugars fed to the protoplasts.
(ii) Isotope effects during the pyruvate dehydrogenase
(PDH) reaction
13 C-depletion in lipids and subsequent 13 C content
in respired CO2 may also result from a kinetic isotope effect during decarboxylation of pyruvate. This
possibility has already been discussed by Park and Epstein (1961), De Niro and Epstein (1977), Rossmann
et al. (1991), Gleixner et al. (1993) and Schmidt and
Gleixner (1998).
Microorganisms provide a simple system to
identify the biochemical steps during which fractionation occurs, leading to 13 C-depletion in lipids (Abelson and Hoering, 1961; De Niro and Epstein, 1977;
Melzer and Schmidt, 1987). Using glucose, pyruvate
or acetate as growth substrate for Escherichia coli,
De Niro and Epstein (1977) showed, as did Abelson and Hoering (1961), little or no fractionation
during glucose metabolism to pyruvate (Embden Meyerhof pathway) but found a substantial fractionation
by about 7–8 during lipid formation from pyruvate.
In addition, positional isotope effects of 1.021 and
1.003 on the C-2 and C-3 of pyruvate, corresponding
to differences in δ 13 C of 18, have been demonstrated using E. coli PDH enzyme in vitro (Melzer and
Schmidt, 1987). As a consequence, acetyl-CoA and
hence fatty acids are 13 C-depleted compared to carbohydrates, as reported above. Similarly, using yeast
pyruvate decarboxylase, De Niro and Epstein (1977)
151
observed a 13 C-depletion in acetaldehyde compared to
supplied pyruvate, which was largely (7.5) confined
to the carbonyl carbon atom of pyruvate (C-2) and only
slightly (1) to the methyl group (C-3).
An additional isotope effect of about 1.009 was
also reported on C-1 of pyruvate (the carbon atom producing CO2 during PDH reaction) using E. coli PDH
(Melzer and Schmidt, 1987). Therefore, CO2 released
during PDH reaction is expected to be 13 C-depleted.
This effect could, however, be masked by the abovementioned 13 C-pattern in the glucose molecules.
De Niro and Epstein (1977) showed experimentally, that the magnitude of the expected isotope
effect depended on the proportion of pyruvate decarboxylated by yeast enzyme in vitro. Since PDH
reaction is one of the several reactions at a branching point, they suggested that other reactions involving
pyruvate as reactant modulate the fraction of pyruvate
oxidised to acetyl-CoA and consequently the isotope
effect during PDH reaction. A similar pattern was also
recently shown by Schmidt’s group (for a review see
Schmidt and Gleixner, 1998). Park and Epstein (1961)
observed a negative correlation between 13 C-depletion
in the lipid fraction and the amount of lipid across
several plant species and algae which they regarded
as consistent with this feature.
Besides, the isotope effect of PDH reaction will
depend on the relative contribution of the enzymesubstrate binding step (i.e., equilibrium isotope effect
thus 13 C accumulation in the enzyme-substrate complex compared to the substrate) and the decarboxylation step itself (i.e., kinetic isotope effect thus 13 Cdepletion in the products). Therefore, the overall isotope effect and thus the released CO2 will have the
signature of the limiting step. A similar pattern (including both rate limitation and corresponding isotope
effects, leading to either enriched or depleted signals) was previously demonstrated for yeast pyruvate
decarboxylase (Jordan et al., 1978).
Accordingly, 13 C-enrichment (or 13 C-depletion) in
respiratory CO2 relative to carbohydrates is expected to be variable among different species, to change
with plant development and with environmental conditions as well as relative activities of different metabolic
pathways.
Metabolic origin of δ 13 C of dark respired CO2 and its
variability
In order to confirm the hypothesis that changes in
metabolic rates may induce changes in signature of
dark respired CO2 , Tcherkez et al. (2003 and unpublished data) conducted experiments on intact leaves
of C3 species under varying leaf temperatures. They
argued, based on the metabolic theory of Park and Epstein (1961), that increasing leaf temperature should
increase the rate of oxidation of lipids rather than
their biosynthesis and, consequently, the light carbons
will be consumed in the Krebs Cycle, so decreasing
the δ 13 C of overall respired CO2 . By connecting a
CO2 -free, closed system, to the GC column of the
elemental analyser, and using a small sample loop,
on-line measurements of δ 13 C of dark respired CO2
were made during changes of temperature on an intact
leaf. Since the specific substrate used for respiration
under varying temperatures was unknown, the overall
respiratory fractionation in the dark was calculated, as
did Farquhar et al. (1989) for the term ‘e’, relative
to the isotopic signature of leaf total organic matter
(see the legend of Figure 2). As expected, for all the
species studied dark respired CO2 was 13 C-enriched
compared to leaf material (i.e., ‘e’ is negative) while
the 13 C content linearly decreased with increasing leaf
temperature, thus with increasing respiration rate (Figure 2). The slope was almost the same for P. vulgaris
and N. sylvestris but different and again more variable
for H. annuus.
One can hypothesise that the changes in δ 13 C of
respired CO2 with increasing leaf temperature observed by Tcherkez et al. (2003) is a direct effect of the
temperature on the temperature-dependent kinetic isotope effect of the PDH reaction. Indeed, a substantial
decrease in the isotope effect with increasing temperature has already been reported during decarboxylation reactions, e.g., a decrease in the isotope effect
by about 4 over the range 15–35 ◦ C during pyruvate
decarboxylation by yeast PDC (De Niro and Epstein,
1977) and by about 10 over the range 25–37 ◦ C
during decarboxylation of arginine by bacterial ADC
(O’Leary, 1980). In fact, as temperature is lowered,
the decarboxylation step becomes more and more rate
limiting and thus the isotope effect of the overall
reaction increases (O’Leary, 1980).
In order to avoid any temperature effect, Tcherkez
et al. (2003) conducted experiments at constant leaf
temperature but under a continuous dark period on intact P. vulgaris leaves. For a given temperature, the
13 C-content in respired CO decreased with the dur2
ation of the dark period, together with a decrease
in leaf carbohydrate content, suggesting that carbohydrate starvation under continuous darkness induced
a switch in the substrate used, from carbohydrates to
152
Figure 3. Relationship between δ 13 C of CO2 respired in the dark
and respiratory quotient (RQ) redrawn from Tcherkez et al. (2003).
Closed symbols were obtained on P. vulgaris intact leaves at either
different leaf temperatures (10, 20 or 30 ◦ C) or at varying dark
period length for a given temperature. Open symbols are from the
literature on different plant species (James, 1953; Park and Epstein,
1961; Smith, 1971). The linear regression does not take into account
data from the literature. The regression equation is: y = 16.57 x −
37.62 (r2 = 0.87).
Figure 2. Relationship between carbon isotope fractionation (e)
during dark respiration and respiration rate in P. vulgaris (A)
N. sylvestris (B) and H. annuus (C) intact leaves under varying leaf
temperature (10, 20 and 30 ◦ C) (Tcherkez and Ghashghaie, unpublished data). ‘e’ is calculated according to Farquhar et al. (1998) as
follows: e = (δ 13 C leaf organic matter – δ 13 C respired CO2 )/(1 +
δ 13 C respired CO2 ).
more 13 C-depleted substrates such as lipids or proteins
(Tcherkez et al., 2003). As it has been earlier proposed
by Smith (1971), this should be confirmed experimentally by measuring the respiratory quotient (RQ). Since
RQ is the ratio of CO2 production to O2 consumption,
it is dependent on the state of oxidation of the substrate used for respiration. Thus from different types of
metabolic oxidation (e.g., lipid or carbohydrate oxidation, or gluconeogenesis) emerge different RQ values
(around 0.6, 1, 0.4, respectively) and a change in RQ
may indicate a switch in respiratory substrate.
Only about 30 years after Smith’s recommendation, Tcherkez et al. (2003) measured simultaneously
isotopic signature of respired CO2 and RQ on P. vulgaris leaves under varying leaf temperatures and under
continuous darkness. Interestingly, they obtained a linear relationship between the two parameters for both
temperature and continuous dark experiments (Figure 3) confirming that changes in the isotopic signature
of respired CO2 originate from substrate switching.
Indeed, RQ values close to 1 indicating highly oxygenated substrates (e.g. carbohydrates) are observed
for a high 13 C content in respired CO2 and RQ values
around 0.6 indicating weakly oxygenated substrates
(e.g., fatty acids) are observed for a low 13 C content
in respired CO2 (Figure 3).
In agreement with the earlier statements of Park
and Epstein (1961) and De Niro and Epstein (1977),
Tcherkez et al. (2003) proposed that there are two
main origins of metabolic CO2 sources: one 13 Cenriched from pyruvate decarboxylation and the other
13 C-depleted from acetyl-CoA degradation through
Krebs cycle. The imbalance between these two
sources may be responsible for the prevalence of 13 C
in overall respired CO2 . Indeed, when carbohydrates
are degraded (RQ around 1) and acetyl-CoA (light carbons) are used for anabolic pathways (e.g., lipid biosynthesis) the isotopic composition of respired CO2
should be close to the mean value of C-3 and C-4 in
glucose molecules (e.g. for the glucose values reported by Rossmann et al., 1999, this would correspond
to −21). By contrast, when lipids are degraded
(RQ around 0.6) acetyl-CoA is produced by oxidation
of fatty acids, thus the isotopic signature of respired
CO2 should be close to the mean value of C-1, C-
153
Figure 4. Predicted δ 13 C of assimilates remaining in the leaf calculated using a net photosynthetic discrimination () of 18 and
a dark respiratory fractionation (e) of 4 for R/A ratios of 0.025
(continuous line), 0.05 (dotted line), 0.1 (dashed line) as a function
of night-length, where R is average night-time respiration rate and
A is average assimilation rate per unit time.
2, C-5 and C-6 of glucose about 6 more negative
than the C-3, C-4 positions. Using a metabolic model,
based only on the 13 C pattern in hexose molecules
(Rossmann et al., 1999) and on the isotope effects of
PDH measured in vitro (Melzer and Schmidt, 1987),
Tcherkez et al. (2003) showed that the observed variation range of δ 13 C of respired CO2 (between −20
and −30) fitted well with the predicted interval.
They concluded that the isotopic signature of dark
respired CO2 in C3 plants is not constant and is determined by (i) the carbon source used for respiration,
i.e., the relative metabolic activities in the cell, (ii)
the non-statistical carbon isotope distribution in glucose molecules and (iii) by possible isotope effects of
respiratory enzymes. Respiratory fractionation in the
wider sense of the definition can be the result of any
of the three processes discussed above.
The studies with concurrent measurement of the
isotopic signature of respired CO2 and the RQ (Tcherkez et al., 2003) showed that a change in respiratory
substrates contributes to the overall dark respiratory
fractionation of leaves under changing temperature regimes and under carbohydrate starvation. However,
the δ 13 C of CO2 respired by leaves in the dark was
also enriched relative to δ 13 C of the main potential respiratory substrates when respiratory CO2 was sampled
in the course of a night of normal length and at
temperatures close to the day-time temperatures (Duranceau et al., 1999; Ghashghaie et al., 2001; Tcherkez
et al., 2003). These results indicate that apparent frac-
tionation relative to the respiratory substrates occurs
frequently in leaves of C3 plants.
In conclusion, it appears that night-time leaf respiratory fractionation will often lead to the release of
carbon that is isotopically heavier than day-time assimilates and shift the overall isotopic signature of
the assimilates remaining in the plant towards more
negative values, in accordance with the model proposed by Henderson et al. (1992). The expected ranges
of this effect are illustrated in Figure 4. On longer
time scales, variation of night-time leaf respiratory
δ 13 C of several per mil can be expected when changes
in environmental conditions or plant ontogeny induce
switching between substrate classes. However, mitochondrial respiration is inhibited in the light even
at light levels as low as 3 µmol Photon m−2 s−1 ).
This light inhibition of mitochondrial respiration was
shown to vary between 16% and 77% (Atkin et al.,
1998, 2000). Therefore the relative effect of respiratory fractionation will differ in the light.
Ultimately, these changes in apparent fractionation will reflect systematic rules associated with positional effects and rate-limiting steps provided that
the biosynthetic pathway is under steady state conditions. Kinetic isotope effects contribute primarily to
isotopic patterns of natural compounds, particularly
for irreversible enzyme steps connected to metabolic
branching events, substrate effects and variations in
metabolic pathway. Increased understanding of the detailed processes involved, and the contributory isotope
signals, will allow prediction and modelling to show
how individual isotope effects are integrated into the
apparent fractionation factor, as measured, and contribute to the overall discrimination expressed during
photosynthetic and respiratory metabolism.
Fractionation during photorespiration
Carbon isotope signature of CO2 produced during
photorespiration and causes of fractionation
Discrimination during photorespiration is primarily
associated with enzymatic fractionation. In the case
of oxygen isotopes, discrimination against the heavier
isotope (18O) can occur during oxygenation by Rubisco, by glycolate oxidase or the Mehler reaction
(Guy et al., 1993). However, the specific effects of
carbon isotope discrimination are found mainly during decarboxylation processes (Jordan et al., 1978;
Ivlev, 2001). Unlike dark respiration, however, there
154
13 C
Figure 5. Schematic diagram of the effects of carbon isotope
discrimination during assimilation and photorespiration in vivo.
are no positional or branch-specific effects, as glycine substrate pools have a high turnover rate (Parnik
et al., 1972) and in vitro studies have demonstrated that
fractionation effects are primarily associated with glycine decarboxylation (Jordan et al., 1978; Ivlev, 2001;
Ivlev et al., 1996, 1999). Therefore, photorespiration
will discriminate against 13 C and photorespired CO2
should, in theory, be depleted in 13 C, leaving the substrate pools enriched. This is analogous to Jordan et al.
(1987), where CO2 liberated during decarboxylation
of pyruvate was depleted in 13 C, which is typical for
such a rate-limiting step, as discussed above. Glycine
decarboxylations in isolated mitochondria from several plant species showed large shifts in the isotopic
composition of CO2 evolved (Ivlev et al., 1996, 1999)
with photorespired CO2 fluctuating between enrichment in 13 C (by up to 8) to being 13 C-depleted by
as much as 16. Again, the extent and direction of
the fractionation effect was highly dependent on reaction conditions, with the variations in fractionation due
to alterations in pH and enzyme co-factors which impacted on the rate-limiting stage of the reaction (Ivlev
et al., 1999; Igamberdiev et al., 2001).
The measurement of leaf 13 C in a glycine decarboxylase (GDC)-deficient barley (Hordeum vulgare L.) mutant has also indicated that photorespiratory fractionation takes place in vivo (Igamberdiev
et al., 2001). That photorespiratory carbon isotope
fractionation leads to production of CO2 depleted in
was supported by the analysis of oxalates, formed
via photorespiration, which were enriched in 13 C
(Raven et al., 1982). Further studies on photorespiratory effects have revealed that net instantaneous discrimination at leaf-level, measured in real time during
gas exchange, is reduced under conditions promoting
increased relative rates of photorespiration (Gillon and
Griffiths, 1997; Gillon, 1997; Lanigan and Griffiths,
unpublished data).
Therefore, the final isotopic composition of CO2
retro-diffusing during gas exchange will be determined by the mixing of both 13 C-depleted CO2 produced from photorespiration with 13 C-enriched CO2
left from carboxylation. In parallel with this, during carboxylation by Rubisco, 13 C-depleted carbon
will contribute to carbohydrate pools, whereas during oxygenation, i.e. the glycolate cycle, and subsequent decarboxylations will result in the formation of 13 C-enriched carbon compounds (Ivlev, 2002,
see Figure 5). Ultimately, if the serine returning as
phosphoglycerate feeds back into the regeneration of
RuBP, then any enrichment in 13 C will be incorporated
into Calvin cycle intermediates.
What is the ‘fractionation factor’ during
photorespiration?
As stated above, net photosynthetic discrimination
has been robustly modelled and takes into account
associated kinetic and enzymatic fractionations, including photorespiratory fractionation (Eq. 1). Thus
we can use the simplified model of Farquhar et al.,
1982 (see Eq. 2) to predict photosynthetic discrimination (i ) as compared to that measured in real-time
during photosynthesis (obs), in CO2 leaving a gas
exchange cuvette. The extent that photorespiratory
fractionation will alter the final net discrimination
value is the product of both the rate of photorespiration (defined by ∗ /Pc ) and the discrimination during
photorespiration (represented by f, the photorespiratory fractionation factor). However, the size of this
fractionation has been the cause for some debate (see
Table 2). Troughton et al. (1974) first directly estimated fractionation associated with photorespiration
(f ) to be between −1.6 and −0.2 by collecting
photorespired air from a stream of CO2 -free air. However, these values for photorespiratory fractionation
were later questioned (Farquhar et al., 1982), since
CO2 leaving the leaf may have been subjected to partial re-assimilation by Rubisco, which would have led
to 13 C enrichment of CO2 .
155
Table 2. Variation in the estimated values of the photorespiratory fractionation factor (f ).
Species
Estimate of f Reference
P. sativum/S. oleracea
−8 to
(mitochondria)
+16
T. aestivum
+2
(minus refixation)
P. vulgaris
+0.5
(minus refixation)
T. aestivum/P. vulgaris
+8
(including refixation)
Glycine max
+7
S. cineraria/S.squalidis +9
S. greyii
+11
Ivlev et al. (1996)
Gillon and Griffiths
(1997)
Gillon and Griffiths
(1997)
Gillon (1997)
Rooney (1988)
Lanigan and Griffiths
(unpublished data)
Lanigan and Griffiths
(unpublished data)
Whether the extent that day respiratory and
photorespiratory (i.e. (photo)respiratory) effects are
manifested in the long-term plant biomass isotopic
composition is debatable, these fractionations have
been shown to comprise a significant component of
net observed instantaneous discrimination (Gillon and
Griffiths, 1997; Gillon et al., 1998). This was first
revealed when i was compared with obs for Piper
aduncum, under high-temperature conditions in the
field in Trinidad. There was a breakdown in the
normal positive correlation between i − obs (the
difference between theoretical photosynthetic discrimination and overall observed discrimination in a gas
exchange system) and increasing A/Pa (assimilation
rate normalised to ambient CO2 partial pressure) (Gillon, 1997; Gillon et al., 1998; Harwood et al. 1998;
Griffiths et al., 1999). This was a direct relationship between ambient temperature and obs during the
measurement periods due to an increase in the rates
of (photo)respiration relative to assimilation, with decreases in obs independent of changes in A/Pa . This
temperature effect was later confirmed for Phaselous
vulgaris; an increase in temperature from 22 ◦ C to
31 ◦ C was observed to elicit a breakdown in the i −
obs versus A/Pa relationship (Lanigan and Griffiths,
unpublished data).
Subsequent studies have attempted to quantify the
effects of photorespiratory fractionation during online discrimination, by estimating the value of f from
the effects on the observed discrimination (obs ) of
retro-diffused CO2 from leaves exposed to different
O2 partial pressures (Gillon and Griffiths, 1997; Gillon, 1997; Lanigan and Griffiths, unpublished data). In
these studies, the discrimination effects of photorespiration (and hence estimation of f ) were assessed
from the effects on the relationship between i −
obs and A/Pa elicited by changing O2 partial pressure (pO2 ). Results are shown for Senecio vulgaris in
Figure 6. Increasing pO2 from 20 mbar to 300 mbar increased photorespiration, as scaled by ∗ (Figure 6A).
When compared to measurements made under nonphotorespiratory conditions (i.e., low O2 partial pressure), i − obs for a given value of A/Pa increased at
higher pO2 (see Figure 6B). This is a result of decreasing obs at higher relative rates of photorespiration,
caused by an increased proportion of 13 C-depleted
CO2 in the total CO2 retro-diffused from the leaf.
Net discrimination (obs ) is the sum of gross
carboxylation, day-time respiration and photorespiration effects. The contribution from day-time respiratory discrimination (the product of appropriate
apparent fractionation factor and rate of day-time respiration -eRd /V c ) and photorespiratory discrimination
(the product of apparent photorespiratory fractionation and rate, - f ∗ /Pc ) can be corrected. This results
in the convergence of the i − obs versus A/Pa
relationships for all pO2 treatments, as discrimination now solely represents photosynthetic fractionation. However, day-time respiratory rate is thought
to be relatively low, so respiratory discrimination
makes only a small contribution to the overall fractionation effects, especially at high assimilation rates
(A/Pa > 1.5 mol m−2 s−1 bar−1 ). Hence, observed
non-photosynthetic fractionation effects are almost
exclusively due to photorespiratory discrimination,
which remains a constant proportion of assimilation.
Therefore, convergence will occur for a given value
of f, since photorespiratory discrimination is defined
as the product of photorespiratory fractionation and
flux (f ∗ /Pa ). Convergence of i − pr versus A/Pa
(where pr is defined as gross photosynthetic discrimination accounting for refixation, see below) is
illustrated for Senecio vulgaris in Figure 6C.
The equations used to account for these (photo)respiratory effects and estimate f are based on the instantaneous discrimination equations of Evans et al. (1986).
Gillon and Griffiths (1997) modified these to include
the above terms for respiratory and photorespiratory
discrimination in order to estimate f for Phaselous
vulgaris and Triticum aestivum. This value was observed to be species-specific (see Table 2). However,
the equation of Gillon and Griffiths (1997) assumed
156
for refixation of a portion of the photorespired CO2
resulted in an increase in the estimates of f for both
species as only a portion of photorespired CO2 was
assumed to leak out of the leaf (Gillon, 1997; Griffiths
et al., 1999, see Table 2). With these assumptions,
values of f were consistent between species and also
agreed with previous estimates made in Glycine max
(Rooney, 1988). Current studies on both shrubby and
herbaceous species of Senecio also observed a 7–9
shift in the estimate of f, depending on how the CO2
fluxes were modelled. Estimates accounting for refixation were similar to those of Gillon (1997) (see
Figure 6C for estimates for S. vulgaris), although values of f were not consistent across species, being
higher for the shrubby species Senecio greyii (Lanigan
and Griffiths, unpublished data).
Ecological implications of fractionation during
dark respiration and photorespiration
Figure 6. The effect of changes in pO2 (oxygen partial pressure) on
gas exchange and isotope discrimination characteristics for Senecio vulgaris (Lanigan and Griffiths, unpublished data). (A), The
compensation point in the absence of respiration ( ∗ ); (B), the relationship between i − obs (the difference between calculated
and measured discrimination) and A/pa (the ratio of assimilation/ambient CO2 partial pressure); (C), the relationship between
i − pr (the difference between calculated and net photosynthetic
discrimination including refixation) and A/pa (the ratio of assimilation/ambient CO2 partial pressure). Symbols denote pO2 treatments
at 20 mbar (grey symbols), 210 mbar (white symbols) and 300 mbar
(black symbols).
total retro-diffusion of all photorespiratory CO2 , so the
fractionation effects observed were assumed to represent those for the total photorespiratory rate. However,
refixation of respiratory CO2 has been demonstrated
to occur in several studies and functions as a means
to maintain an electron sink under adverse conditions
(Gerbaud and Andre, 1987; Bort et al., 1996; Loreto
et al., 2001). Hence, further modifications to account
Respiration is a key component of net ecosystem and
global gas exchange, with much effort currently being
directed towards resolving CO2 fluxes, and specifically regional carbon sinks (Ciais et al., 1995a; Yakir
and Wang, 1996; Buchmann et al., 1998; Ciais and
Meijer, 1998; Ciais et al., 1999; Bowling et al., 2001).
These estimates are dependent on measurements of
ecosystem discrimination (E ) as well as regional
[CO2 ] in order to determine the magnitude of the
terrestrial carbon sinks. In turn, ecosystem discrimination (E ) represents the net effect of both vegetation
and soil processes over an integrated period of time
and is defined (Lloyd and Farquhar 1994) as:
E =
δ 13 CT − δ 13 CR
,
1 + δ 13 CR
(3)
where δ 13 CT and δ 13 CR are the isotopic composition
of tropospheric and ecosystem-respired CO2 . Keeling (1958) predicted that the integrated δ 13 CR of all
respiring components could be estimated via the intercept of the regression of δ 13 CR and 1/[CO2 ]. However,
the extent to which δ 13 CR varies both temporally and
spatially is poorly understood and this could alter conclusions about the nature of the terrestrial carbon sink
(Bowling et al., 2002). In particular, the soil respiratory component of δ 13 CR requires further study.
While the largest variations in δ 13 CR are mainly due
to the dominant vegetation type (Buchmann et al.,
1998), other causal factors can induce spatial and
temporal variations in δ 13 CR of up to 10 within
157
pure C3 stands (Buchmann et al., 1997). Site history must be taken into account if there has been a
transition between C3 and C4 species growing on the
area in question (Buchmann et al., 1998). Changing
environmental conditions have been shown to cause
large variations in δ 13 CR via effects on assimilation
rates and stomatal conductance. Ekblad and Hogberg
(2001) demonstrated that alterations in assimilation
rate caused by changing air humidity were linked to
changes in δ 13 CR that occurred one to four days later
and comprised up to 65% of the soil respiration signal,
while Bowling et al. (2002) have also found a strong
link between δ 13 CR and changes in vapour pressure
deficit.
However, respiratory fractionation effects between
canopy and soil are debatable. Fractionation due to
microbial respiration was initially believed to be significant (Blair et al., 1985), although recent studies have
estimated rhizosphere respiratory fractionation at less
than 1 (Ekblad and Hogberg, 2000; Ekblad et al.,
2002). It is not clear whether the respiratory fractionations described in the previous sections translate
into a significant component at canopy and ecosystem
level, but distinguishing between autotrophic and heterotrophic contributions to soil respiration is a key aim
at present. Current approaches using flux partitioning
of 13 CO2 have discounted any fractionation associated with respiration per se (Bowling et al., 2001).
Additional measurements are required to determine
whether the fractionations described for P. vulgaris,
N. tabacum and H. annuus (Duranceau et al., 1999;
Ghashghaie et al., 2001, Tcherkez et al., 2003) alters
soil respiratory efflux, particularly those associated
with changing environmental conditions and whether
this affects NEE partitioning.
Finally, we note that whilst the 18 O signal in CO2
does provide a more definitive means to separate soil
from canopy-based gas exchange processes (due to the
evaporative enrichment in leaves), this technique has
not yet been widely applied at canopy and ecosystem
scales, in contrast to global modelling (Ciais et al.,
1995a, b, 1999; Buchmann et al., 1997; Brooks et al.,
1997).
The effect of photorespiratory discrimination on
the overall carbon isotope signature of plant biomass
is unclear. Ivlev et al. (1999) has suggested that isotopic differences in δ 13 C of leaves, stems and seeds
of Triticum aestivum are due to differences in the relative contribution of photorespiration at the stages
of organ formation. Also, leaves of GDC-deficient
Hordeum vulgare have been observed to be enriched
in 13 C relative to wild-type plants (Igamberdiev et al.,
2001). However, photorespiratory processes are generally transient and during leaf expansion very little
structural carbon will be photorespiratory in origin. As
a result, it is unlikely that photorespiratory discrimination contributes significantly to variation in the carbon
isotopic composition of plant biomass.
Finally the fractionation associated with respiration in the light (Rd ) is unknown. This light-mediated
inhibition is thought to be due to rapid light inactivation of key enzymes such as pyruvate dehydrogenase
complex and NAD+ -malic enzyme (Hill and Bryce,
1992). While fractionations associated with Rd will
have a negligible effect on organic matter or carbohydrate carbon isotope composition, there may be
detectable effects in retro-diffused CO2 , which will
vary depending on species and environmental conditions. In studies on snow gum, for instance, temperature has been shown to have a large effect, invoking
high levels of inhibition (97%) at 30 ◦ C and high
irradiance, but with inhibition being almost totally alleviated under low (6 ◦ C) temperatures (Atkin et al.,
2000). Teasing apart the competing fractionations associated with photorespiration and light respiration
and, hence, obtaining a value for e in the light is technically extremely difficult however, and little work has
been done to address this problem.
Photorespiratory fractionation does make a significant contribution to net instantaneous discrimination.
Therefore, in order to interpret day-time Keeling plots,
and thus deconvolute day-time carbon fluxes, the contribution of the photorespiratory component will need
to be assessed. This would then enable researchers to
scale up changes in atmospheric CO2 temporally from
the current daily assessments to changes occurring
across a whole season.
Conclusions
Whilst the more complicated model of discrimination
(Eq. 1) accounts for the effects of fractionation during
(photo)respiration on resultant plant organic material,
there are several important points to be considered.
Firstly, there is uncertainty as to the magnitude and
definition of the specific fractionation factors e and f ;
secondly, any apparent fractionation expressed during
photosynthesis or dark respiration may vary with environmental conditions, and thirdly, the impact of such
fractionations may be significant in real time as we try
158
to assess (photo)respiratory exchanges in the global
context of climatic change.
We have used a variety of techniques to resolve
the magnitude of fractionation processes, from direct isotopic analysis of released CO2 (and associated
mass balance effects on residual C pools) to modelling
the discrimination effects instantaneously during gas
exchange. In general terms, fractionation during consumption of sucrose produces CO2 which is enriched
in 13 C by some 6, such that the fractionation factor
e = −6, meanwhile, fractionation expressed during
photorespiration tends to oppose this process, with
CO2 released being depleted in 13 C (i.e., f = +8 to
+11).
However, overall fractionation during dark respiration is dependent on species and environmental
conditions. The value expressed reflects a combination
of three processes, namely the carbon source being
utilised, specific positional effects as well as additional enzymatic fractionation. For photorespiration,
the fractionation factor is to some degree speciesspecific, but the value expressed is also dependent on
stomatal conductance and the magnitude of refixation
of photorespired CO2 . Indeed, we should conclude
that in contrast to specific fractionation factors which
can be allocated to diffusion and dissolution of CO2 ,
or carboxylation by Rubisco or PEPC (see discussion
of Eqs 1 and 2 above), the apparent fractionation for
respiration and photorespiration are variable. Thus it is
intriguing that temperature does not alter the fractionation expressed by Rubisco (O’Leary, 1981), allowing
one fairly consistent value to be applied throughout.
During dark respiration, there are interactions between
specific enzymatic isotope effects for multiple reactions and source-specific substrate effects, which are
also dependent on temperature and other environmental constraints. Thus, we now define the resultant
‘apparent’ fractionation to represent a combination of
these interacting processes.
In a similar way, the apparent fractionation
expressed during photorespiration in not simply
a product of the isotope effect for glycine decarboxylase, determined in vitro. How important are
these fractionations quantitatively? There is often an
excellent agreement between gas exchange-derived
measurements of Pi /Pa and the predicted or measured
organic material carbon isotope composition (using
Eq. 2). Indeed, this may in part reflect the way that
respiratory and photorespiratory fractionations would
effectively cancel each other out, provided that the
respective isofluxes (i.e. sum of fractionation and
associated gaseous flux) were quantitatively similar.
However, the use of the isoflux terminology brings
us to one of the most important current applications
of stable isotopes, namely in trying to resolve the
contrasting interplay between photosynthetic and respiratory fluxes at the global level. Here, understanding
CO2 exchanges at leaf, soil and canopy level and their
relationship with the inter-annual changes seen seasonally at the global level (particularly in the northern
hemisphere), are dependent on resolving respiratory
inputs across each of these levels.
We now need to conduct additional measurements
to determine the way that respiratory and photorespiratory overall fractionations translate into altered
organic carbon isotope signals in plant material, particularly under elevated CO2 for the future, or the high
photorespiratory conditions pertaining at the last glacial maximum, when CO2 levels were much lower
than at present. Additionally, the way that fractionation during dark respiration could alter carbon isotope
signal of carbon exported from the canopy, when released some four or five days later by roots, is another
fundamental application requiring additional experimentation. In conclusion, the provision of more definitive values for fractionation factors associated with
(photo)respiration and dark respiration, as described in
this review, will allow us to explore their implications
at global levels, whilst additional work is required
to resolve the interplay between compound-specific
positional effects and fractionation at the enzymatic,
biochemical and molecular levels.
Acknowledgements
Gary Lanigan and Salvador Nogués acknowledge
the financial support provided through the European
Community’s Human Potential Program under contract HPRN-CT-1999-00059, NETCARB. We also acknowledge the referees for their valuable contribution
to the drafting of this paper.
Appendix
Definitions
Isotope ratio: R is defined as the molar ratio of the
heavy to light isotope e.g. for carbon, R = 13 C/12 C.
Isotope effects: When physical and chemical processes modify the isotope ratio of product compared
to substrate (source), there is either an enrichment or
depletion of the heavier isotope.
Kinetic isotope effect: For irreversible reactions the
kinetic isotope effect, αk , is defined as the ratio of the
159
rate constants for the molecules containing
(k12 and k13 , respectively) as follows:
12 C
and
13 C
αk = k 12 /k 13
(A1)
For CO2 diffusing in air, α is 1.0044. When the
source is large enough to be not appreciably affected
by product formation, then the kinetic isotope effect
is:
αk, = Rs /Rp
(A2)
where, Rs and Rp are the isotope ratios of the source
and of the product.
Equilibrium isotope effect: For reversible reactions,
an equilibrium isotope effect, (αeq ) is the ratio of equilibrium constants for the compounds containing 12 C
and 13 C (K12 and K13 , respectively) as follows:
αeq = K 12 /K 13
(A3)
Fractionation factor: Because isotope effects are very
small, it is easier to describe each transformation involving isotopes as a fractionation factor, (1 − α), and
express in parts per thousand (). For example, the
fractionation factor associated with CO2 diffusing in
air, a, which depletes the heavy isotope, is +4.4.
Limiting step: When enzymatic reactions proceed by
multi-step mechanisms the overall isotope effect depends on both the ‘intrinsic isotope effect’ of the
isotopically sensitive step and on the extent to which
this step is rate-limiting (O’Leary et al., 1992).
Branching point: When a substrate contributes to
other competing reactions the overall isotope effect
will depend on the relative contribution of the substrate to the enzymatic reaction and to the other
reactions.
Positional effect: Carbon atoms in a particular molecule can show large differences in isotope ratio,
dependent on the isotope effects associated with each
biosynthetic process.
Temperature dependence of isotope effects: Isotope
effects of enzymatic reactions depend on physical and
chemical conditions with some decarboxylation steps
less rate-limiting with increasing temperature or because of pH modification with temperature (O’Leary,
1980).
Isotope discrimination: We define the overall shift
in isotope composition from source to product as
discrimination (), expressed as
= α − 1 = (Rs /Rp ) − 1.
(A4)
According to Eq. A4, to determine the carbon isotope
discrimination by plants, one should measure the carbon isotope ratios in the air (source) and in the plant
(product), Ra and Rp , but in practice each is measured
against a defined standard, with an isotope ratio arbitrarily set to 0. The carbon isotope composition of
plant material, δ 13 Cp (expressed in ), is defined as
δ 13 Cp = (Rp − Rstd )/Rstd = (Rp /Rstd ) − 1, (A5)
where, Rstd is the (molar) isotope ratio, 13 C/12 C, of
the standard. Using Eq. A4 and Eq. A5, the overall
carbon isotope discrimination, , could be calculated
as follows:
= (δ 13 Ca − δ 13 Cp )/(1 + δ 13 Cp )
(A6)
δ 13 Ca
where,
is the carbon isotope composition of air.
Atmospheric CO2 has an isotope ratio of approximately −8, and typical C3 plant material −28, and
using Eq. A6, an overall discrimination () value of
20.6 (Farquhar et al., 1989). Discrimination can
be approximated as the difference between the carbon isotope composition of the source and that of the
product:
= δ 13 Ca − δ 13 Cp
(A7)
In this case, the discrimination value for C3 plants
on the basis of plant organic matter will be approximated to = (−0.008) − (−0.028) = 20.
Apparent fractionation: The fractionation effects for
respiration or photorespiration (respectively termed e
and f ) represent many biochemical reactions. Whilst
these can be measured as the difference between
organic material and CO2 released (as an overall
discrimination: see Eq. A7), we have adopted the
term ‘apparent fractionation’ to distinguish between
individual fractionation factors and the cumulative
fractionation which is measured for a given process.
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