Carbon Isotopic Composition of C3 and C4 Plants
JM Saquing and G Sinclair
Introduction
Variations in the isotopic composition in elements like carbon are used to understand the
dynamics of natural processes that include geology, chemistry, biology, and ecology (O’Leary et
al. 1982).
One of the most standard comparisons of differences in isotopic ratios is the
comparison of 13C to 12C in plants to determine photosynthetic pathway of plants. This section
reviews how the difference in carbon fractionation is used to predict the photosynthetic pathway
of C3 and C4 plants.
The objectives of this section are:
 Examine the morphology and physiology C3 and C4 in relation to their environment
 To examine the factors controlling the δ13C values of C3 and C4 plants
 To determine whether the plants identified in the study have C3 or C4 metabolism
Environmental impacts on plant morphology and physiology
Plants interact with the environment differently depending on their morphology and physiology.
C3 plants are relatively inefficient in using CO2 and have their photosynthetic apparatus in the
outer mesophyll cells. To compensate for this inefficiency stomata must remain open longer
exposing them to potentially increased evapotranspiration and respiration rates. As a result these
plants grow better in cooler moist environments with elevated CO2 concentrations. The enzymes
of C4 plants located in the mesophyll are more efficient in fixing CO2 which decreases the time
stomata must remain open and decreases the evapotranspiration and respiration rates compared
to C3 plants. Consequently, C4 plants are better adapted to warmer and dryer environments.
This is depicted in the Figure 1, below, taken from Ehlinger 1997.
Figure 1. Atmospheric CO2 vs daytime growing temperature. C4 plants are favored in warm climates
with less CO2 while C3 plants are favored in cooler climates with more CO2. Intermediate climates do not
confer a distinct advantage to either photosynthetic pathway (Ehlinger 1997).
The following section will more clearly delineate the mechanisms behind these environmental
adaptations. One important consequence behind these physiological differences is that plants are
different in how they fractionate the carbon isotope of atmospheric CO2. The carbon isotopic
signatures may be used to quickly evaluate plant physiology that provides insight into their
ecology.
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C3 and C4 Leaf Anatomy and Photosynthesis
C3 and C4 leaves have both mesophyll cells containing chloroplasts (Figure 2). The main
difference between C3 and C4 leaves is the presence of bundle sheath cells which also contain
chloroplasts. This difference in leaf structure affects the diffusion and fixation of CO2 in both
plants.
Figure 2. Leaf structure of C3 and C4 plants (http://staff.science.uva.nl/~bjansen/research.html)
C3 plants derive their name from the first stable carbon compound produced after carbon fixation
which is a 3 carbon molecule called phosphoglyceric acid (PGA). In C3 plants, CO2 is fixed
within the mesophyll cells through the Calvin cycle (Figure 3). CO2 is reacted with ribulose
biphosphate (RuBP) by the enzyme ribulose biphosphate carboxylase/oxygenase (RuBisCO).
RuBisCO.
RuBisCO is an inefficient enzyme with low substrate specificity (i.e. sometimes
fixes O2 instead of CO2).
It preferentially fixes
12
CO2 over
13
CO2, resulting in isotope
fractionation during carboxylation (Griffith, 2006).
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Figure 2. Calvin Cycle (Wikepedia.com)
The first carbon compound produce in C4 plants is a 4 carbon molecule (i.e. oxaloacetate; Figure
4). C4 plants follow the Hatch-Slack Pathway wherein CO2 is first incorporated through the
carboxylation of phosphoenolpyruvate (PEP) by the enzyme phosphoenol-pyruvate carboxylase
(PEP carboxylase) in the mesophyll cells. PEP carboxylase is a more efficient enzyme than
RuBisCO and accounts for the environmental tolerances described in the first section.
Phosphoenol-pyruvate carboxylase also does not use CO2 (g) as a substrate as does RubisCo but
uses bicarbonate, HCO3-. Bicarbonate is formed when dissolved CO2 reacts with water. The C4
acids that are produced from the carboxylation of PEP are transported to the bundle sheath where
CO2 is re-released and then fixed again through Calvin Cycle.
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Figure 4. Hatch-Slack Pathway (Wikepedia.com)
Isotopic Fractionation
The carbon isotopic composition of plants are primarily influenced by the isotopic composition
of the CO2 source, isotopic fractionation resulting from CO2 fixation and the isotopic
composition and quantity of CO2 lost through respiratory processes (O’Leary,1980). C3 and C4
plants have different δ13C values, -28.1±2.5 ‰, -13.5±1.5 ‰ respectively (Troughton et al.
1975). Among C3 and C4 plants, δ13C variation can range from 2-5‰.
The first fractionation of the heavy and light CO2 is by a simple diffusion. The difference of
diffusion rates between the light isotope (12C), which moves a little faster through air than the
heavy isotope (13C), is a kinetic effect. The primary difference in isotopic composition is due to
the isotopic fractionation of the heavy (13C) and light (12C) isotopes by the biological processes
of CO2 fixed (C gained) and respiration (C lost) (O’Leary,1980 ).
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C3 Plants
Farquhar et al (1982) developed and validated a model that describes the fractionation of carbon
isotopes during C3 photosynthesis.

13
C plant  
13
pa  pi
pi
Cenv  a
b3
pa
pa
Where
pi
, ratio of intercellular and atmospheric partial pressures of CO2
pa
 1 C env , composition (‰) of CO2 in the environment (i.e. -7.7 ‰, atmosphere)
3
a , diffusion isotope effect (4.4 ‰ in air)
b3, RuBisCO isotope effect (30‰, corrected for equilibrium effect on CO2 dissolution and
reaction with H2O)
This model shows that if stomata are always open, CO2 can freely diffused in and out, pa ≈ pi
then δ13Cplants ≈ -37‰. In this case, RuBisCO can be selective against
13
CO2. If stomata is
normally closed, pa >>> pi then δ13Cplants ≈ -11.4‰. Because of the limited exchange of CO2
between the leaf and the atmosphere, RubisCO will be forced to use whatever the isotopic
composition of CO2 inside the leaf. Since 12CO2 diffuse faster than 13CO2, then δ13C plants will
be more 13C enriched, closer to atmospheric value. Thus, C3 plants tend to have values more in
the range closer to -37‰.
C4 Plants
For C4 photosynthesis, the basis for discrimination will be more complicated due to different
leaf structure and metabolic pathway. First, the dissolution of CO2 and conversion to HCO3- (a
chemical process driven by equilibrium) results in a thermodynamic effect in the isotopic
fractionation of the heavy and light isotope (-9.0 ‰, Mook et al. 1974).
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(O’Leary, 1992)
This thermodynamic isotope effect (Farquhar 1989) is followed by a kinetic isotope effect by
the enzyme that catalyzes fixation of bicarbonate to PEP. This enzyme, PEP carboxylase is very
efficient and binds to HCO3- better than RubisCo does to CO2 in that it does not easily lose
HCO3-. Since the bicarbonate used in the initial carboxylation is relatively enriched in
13
C, the
CO2 released from C4 acids and subsequently fixed through Calvin cycle in the bundle sheath
would be relatively enriched in
13
C compared to CO2 fixed in C3 plants. As a result, C4 plants
are more enriched in 13C than C3 plants.
Farquhar et al. (1983) revised the C3 model to describe isotope discrimination in C4
photosynthesis:

13
C plant  
13
Cenv  a
pa  pi
p
 (b 4  b 3 ) i
pa
pa
where,
pi
, ratio of intercellular and atmospheric partial pressures of CO2
pa
 1 C env , composition (‰) of CO2 in the environment (i.e. -7.7 ‰, atmosphere)
3
a , diffusion isotope effect (4.4 ‰ in air)
b3, RuBisCO isotope effect (30‰, corrected for equilibrium effect on CO2 dissolution)
b4 Fractionation of PEP carboxylase -5.7
Φ Leakiness factor of 0.37 which decreases the fractionation attributed to Rubisco
In C4 plants CO2 enters the stomata and dissolves in the mesophyll and converted to HCO3which, in equilibrium with CO2 is enriched in 13C. HCO3- is fixed by PEP carboxylase and then
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enters the bundle sheath cell where CO2 is refixed with Rubisco. If the bundle sheath was gas
tight there would be no further fractionation because there are not any biochemical branchs
which allow for fractionation so Rubisco uses the C12 and C13 similarly as it enters the bundle
sheath. However, there is leakage from the bundle sheath back to the mesophyll. This leakage
(accounted for by Farquhar 1989 as Φ) permits some fractionation by the Rubisco in the bundle
sheath (according to Farquhar 1989) making the δ13C value more negative than would be
predicted strictly from the fractionation of CO2 atm by PEP carboxylase.
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Rubisco in the bundle sheath (according to Farquhar 1989) making the δ13C value more negative
than would be predicted strictly from the fractionation of CO2 atm by PEP carboxylase.
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Metabolism of Plants in Prairie Ridge Study
Based on the δ13C values of leaf biomass, three of the plants identified have C3 metabolism and
one is considered a C4 plant (Table 1.) Identification of photosynthetic pathway was based
solely on the δ13C values of leaf since this is the organ primarily responsible for photosynthesis.
Furthermore, most studies classified C3 and C4 plants based on the δ13C values of leaf biomass
(citations in Farquhar et al., 1989, Hattersley, 1982, Buchmann et al., 1996, Still et al., 2003).
Figure 5 shows the δ13C values of all samples (i.e. include leaf, stem and root) taken for each
plant identified in the study plot.
Table 1. δ13C values of leaf and stem sub-samples and metabolic classification of plants
identified from Prairie Ridge.
Metabolic
Classification
Sample ID
Identity
δ13C
‰
C3
C3
C3
C3
PRP-12-brown blade
PRP-9-brown blade
PRP-9-green blade
PRP-8-leaf
C3
PRP-3-leaf
Fescue
Fescue
Fescue
Horsenettle
Spanish needles
C3
Average
Stdev
% var
PRP-4-leaf
Spanish needles
C4
C4
C4
C4
C4
C4
C4
C4
Average
Stdev
% var
PRP-11-brown
PRP-11-green
PRP-6-brown blade
PRP-6-green blade
PRP-6-stem
PRP-13-brown leaf
PRP-14 green
PRP-14-brown
Bermuda Grass
Bermuda Grass
Bermuda Grass
Bermuda Grass
Bermuda Grass
Bermuda Grass
Bermuda Grass
Bermuda Grass
-28.63
-29.90
-29.35
-30.29
-31.53
-31.61
-30.22
1.19
3.93
-13.63
-12.47
-13.58
-13.28
-12.41
-13.82
-13.89
-13.17
-13.28
0.57
4.32
Among the plants, Horsenettle, ragweed/bidens or Spanish needle, fescue, and Bermuda grass,
the Bermuda grass was the only C4 plant identified.
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13C
-30
Fescue
Horsenettle
Ragweed
Bermuda grass
-20
-10
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Figure 5. Preliminary C3 vs C4 designation based on both δ13C signature and plant ID.
Brief Discussion and Summary
The δ13C signatures of these plants are consistent and within the range of what we would expect
for C3 and C4 plants. The measured δ13C values among C3 plants vary by 1.2 ‰, which is
within the reported interspecies variation for C3 plants, 2-5 ‰ (O’Leary, 1981). Similar variation
is obtained for different leaf samples of the Bermuda grass. Bermuda samples were taken from
different location within the study plot. Troughton (1974) reported intra-species variation of δ13C
up to 3‰. The difference in δ13C between C3 and C4 plants, ~ 17‰ is comparable to the carbon
isotope offset observed in tallgrass prairie, ~ 16‰ (Still et al. 2003).
Anomalous values (Figure 5: low values in the ragweed) may be attributed to different parts
(roots vs. leaves) and will be discussed by another group. The solitary low δ13C value in the
Bermuda grass however does not seem to be an intermediate value as one would see in different
plant parts and is most likely a mislabeled sample (Hobbie, 2003).
Summary of Factors Controlling Isotopic Composition of Plants

Isotopic Composition of CO2 source

Kinetic Effects
o Diffusion
o Enzyme fractionation
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
Thermodynamic effects (equilibrium processes)

Temperature, humidity, light, fertilizer (Farquhar 1989)
Because our study site was 1 m2 without any other plots for comparison, we have ignored many
of the other environmental factors like CO2 source, light, humidity, temperature, and fertilizer
that can also impact the fractionation of CO2 assuming these factors were not significantly
different between the plants.
In general the plot has been recently farmed so it is difficult to comment on the species
composition of the site. North Carolina does have an intermediate climate so the composition of
C3 vs C4 plants does not seem that surprising. Similarly, since this site is so recently disturbed it
is difficult to compare with other prairie systems (e.g. Archer 1984 and Polly 2002).
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