Journal of Experimental Botany, Vol. 59, No. 7, pp. 1695–1703, 2008
doi:10.1093/jxb/ern001 Advance Access publication 28 March, 2008
SPECIAL ISSUE RESEARCH PAPER
C4 photosynthetic isotope exchange in NAD-ME- and
NADP-ME-type grasses
Asaph B. Cousins1,2,*, Murray R. Badger1,2 and Susanne von Caemmerer1
1
Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University,
Canberra, Australian Capital Territory, 2601 Australia
2
ARC CoE, Plant Energy Biology, Research School of Biological Sciences, Australian National University,
Canberra, Australian Capital Territory, 2601 Australia
Received 27 September 2007; Revised 19 December 2007; Accepted 21 December 2007
Abstract
Monitoring photosynthetic isotope exchange is an
important tool for predicting the influence of plant
communities on the global carbon cycle in response to
climate change. C4 grasses play an important role in
the global carbon cycle, but their contribution to the
isotopic composition of atmospheric CO2 is not well
understood. Instantaneous measurements of 13CO2
(D13C) and C18OO (D18O) isotope exchange in five
NAD-ME and seven NADP-ME C4 grasses have been
conducted to investigate the difference in photosynthetic CO2 isotopic fractionation in these subgroups.
As previously reported, the isotope composition of the
leaf material (d13C) was depleted in 13C in the NAD-ME
compared with the NADP-ME grasses. However, D13C
was not different between subtypes at high light, and,
although D13C increased at low light, it did so similarly
in both subtypes. This suggests that differences in leaf
d13C between the C4 subtypes are not caused by
photosynthetic isotope fractionation and leaf d13C is
not a good indicator of bundle sheath leakiness.
Additionally, low carbonic anhydrase (CA) in C4
grasses may influences D13C and should be considered when estimating the contribution of C4 grasses to
the global isotopic signature of atmospheric CO2. It
was found that measured D18O values were lower than
those predicted from leaf CA activities and D18O was
similar in all species measured. The D18O in these C4
grasses is similar to low D18O previously measured in
C4 dicots which contain 2.5 times the leaf CA activity,
suggesting that leaf CA activity is not a predictor of
D18O in C4 plants.
Key words: C4 grasses, carbonic anhydrase, isotope
discrimination, leakiness.
Introduction
Isotope analysis of atmospheric carbon dioxide (CO2) is
an important tool for monitoring changes in the global
exchange of CO2 (Flanagan and Ehleringer, 1998; Yakir
and Sternberg, 2000). Leaf-level models of carbon isotope
exchange (D13C) and oxygen isotope exchange (D18O) in
C4 plants are used to help interpret the response of C4
plants to changing environmental conditions as well as
predict the contribution of C4-dominated ecosystems to the
global carbon cycle. However, to interpret the atmospheric
CO2 isotopic signature requires an understanding of the
isotopic fractionation steps associated with specific processes during leaf gas exchange (Yakir and Sternberg,
2000). Additionally, it remains unclear how photosynthetic isotope discrimination differs between the two major
C4 biochemical subtypes; the NAD malic enzyme (NADME) and the NADP malic enzyme (NADP-ME) which
differ in their biochemistry, leaf anatomy, and geographic
distribution (Hattersley, 1983; Hatch, 1987).
Most C4 plants utilize a compartmentalized CO2concentrating mechanism between the mesophyll and
bundle sheath cells (BSCs) to increase the CO2 partial
pressure (pCO2) around the site of ribulose-bisphosphate
carboxylase/oxygenase (Rubisco) in the BSC. The specialized biochemistry and leaf anatomy of C4 plants result
in a pCO2 around the site of Rubisco several fold higher
than current atmospheric levels, significantly reducing the
* Present address and to whom correspondence should be sent: School of Biological Sciences, Washington State University, Pullman, WA 99164-4236,
USA. E-mail: [email protected]
ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1696 Cousins et al.
rates of photorespiration and consequently increasing their
photosynthetic efficiency, particularly under high light and
temperature (Hatch, 1987; Kanai and Edwards, 1999). The
dominance of C4 plants in open, hot, and arid environments is largely related to the enhanced photosynthetic
efficiency of C4 plants compared with C3 plants under
these environmental conditions (Osmond et al., 1982;
Pearcy and Ehleringer, 1984; Long, 1999; Ghannoum
et al., 2001). Within the C4 species, the geographic
distribution of the subtypes differ with the occurrence of
NADP-ME, increasing with increases in the average
annual rainfall and the NAD-ME decreasing as a percentage of total C4 grasses (Teeri and Stowe, 1976; Hattersley,
1983; Sage, 2001). Traditionally, the difference in geographic distribution between the two C4 subtypes has been
linked to differences in physiology, primarily the difference in bundle sheath leakiness [/: defined as the fraction
of CO2 fixed by phosphoenolpyruvate carboxylase
(PEPC) that subsequently leaks out of the BSCs] as
estimated from the carbon isotope composition of leaf
material (d13C) (Hattersley, 1982; Farquhar, 1983). However, there are numerous factors which influence d13C in
C4 leaves, including the availability of intercellular CO2,
temperature, light intensity, and post-photosynthetic fractionation, and Henderson et al. (1992) found no correlation between d13C of leaf material and the short-term
measurements of D13C in 11 C4 species.
The differences in D18O between photosynthetic types
may help distinguish the contribution of the influence of
C3 versus C4 on the global CO2 cycle. The d18O of the
H2O at the site of CO2–H2O oxygen exchange within
a leaf (dex) is the primary factor influencing the d18O of
CO2 diffusing out of a leaf (Farquhar et al., 1993). The
proportion of CO2 in isotopic equilibrium with the H2O at
the site of oxygen exchange (h) will also influence the
d18O of the CO2. The value of h is determined by the
balance between the gross flux of CO2 into the leaf and
the activity of CA at the site of CO2–H2O oxygen isotope
exchange as this influences the residence time of CO2
within a leaf and thus the number of hydration reactions
per CO2 molecule (Gillon and Yakir, 2000a, b). Measurements of D18O during leaf gas exchange have shown
D18O to be much lower in two NADP-ME C4 monocots
(Zea mays and Sorghum bicolor) than what is commonly
observed for C3 species (Gillon and Yakir, 2000a, b). A
survey of leaf CA activity in species belonging to
different photosynthetic functional types found that the
C4 monocots in general had relatively low CA content
(Gillon and Yakir, 2001). It has also been shown that
D18O is sensitive to relatively small changes in CA
activity due to an antisense silencing in the C4 dicot
Flaveria bidentis even when rates of photosynthesis were
unaltered (Cousins et al., 2006b).
In this study, five NAD-ME- and seven NADP-ME-type
C4 grass species, grown under common growth condi-
tions, were used to address three distinct questions: (i)
whether short-term online measurements of D13C were
different between the two subtypes as generally seen in the
leaf d13C signature; (ii) whether D18O was different between
the two subtypes; and (iii) whether CA activity was
different between the subtypes and accurately predicted the
measured isotopic equilibrium of CO2 and leaf H2O.
Materials and methods
Growth conditions
Plants were grown during the summer months in a glasshouse under
natural light conditions (27 C day and 18 C night temperatures).
Twelve C4 grass species, five from the NAD-ME and seven from
the NADP-ME subtypes (Table 1), were grown in 5.0 l pots in
garden mix with 2.4–4 g of Osmocote/l of soil (15/4.8/10.8/1.2 N/
P/K/Mg+trace elements: B, Cu, Fe, Mn, Mo, Zn; Scotts Australia
Pty Ltd, Castle Hill, Australia) and watered daily. There were 3–4
replicate pots per species and each pot contained 2–3 individuals of
the same species.
Online 13CO2 and C18OO discrimination
The uppermost fully expanded leaves were placed into the 6 cm2
leaf chamber of the LI-6400 portable gas-exchange system (Li-Cor,
Lincoln, NE, USA) and equilibrated under measurement conditions
for a minimum of 1.5 h (Cousins et al., 2006a, b). Air entering the
leaf chamber was prepared by using mass flow controllers (MKS
instruments, Wilmington, MA, USA) to obtain a gas mix of
909 mbar dry N2 and 48 mbar O2 which contained no water vapour.
A portion of the nitrogen/oxygen air was used to zero the mass
spectrometer to correct for N2O and other contaminants contributing
to the 44, 45, and 46 peaks. Pure CO2 (d13CVPDB ¼ –29&, and
d18OVSMOW¼24&) was added to the remaining air stream to obtain
a CO2 partial pressure of ;531 lbar. Low oxygen (48 mbar) was
used to minimize contamination of the 46 (m/z) signal caused by
the interaction of O2 and N2 to produce NO2 with the mass
spectrometer source. Simultaneous measurements of leaf gas
exchange and isotope discrimination were determined by linking
the LI-6400 gas exchange system to a mass spectrometer through an
ethanol/dry ice water trap and a thin, gas-permeable silicone
membrane (Cousins et al., 2006a, b, 2007; Griffiths et al., 2007).
Calculations of 13CO2 discrimination
The model of C4 carbon isotope discrimination (D13C) from
Farquhar (1983) was used to determine which factors in the model
would influence D13C consistent with the experimental data. The
simplified model predicts that
D13 C ¼ a þ ðb4 þ ðb3 sÞ 3 / aÞ 3 pi =pa
ð1Þ
where pa and pi represent the pCO2 of the air surrounding the leaf
and in the intercellular air spaces, respectively; a (4.4&) is the
fractionation during diffusion of CO2 in air; s (1.8&) is the fractionation during CO2 leakage from the BSCs; and the fractionation
by Rubisco is b3¼30& (Roeske and Oleary, 1984). The combined
fractionation of PEPC and the isotopic equilibrium during dissolution of CO2 and conversion to bicarbonate (b4) was calculated as
(Farquhar, 1983)
b4 ¼ 5:7 þ 7:9Vp =Vh
ð2Þ
HCO–3
are not at
indicating that the fractionation when CO2 and
equilibrium depends on the rate of CO2 hydration (Vh) and the rate
Isotope discrimination in C4 grasses 1697
Table 1. Net CO2 assimilation and carbon isotope discrimination
List of NAD-ME and NADP-ME C4 species used in this study, net CO2 assimilation rates, the ratio of intercellular to atmospheric CO2 partial
pressure (pi/pa), online carbon isotope discrimination (D13C), and the isotopic composition of leaf material (d13C) measured in the youngest fully
expanded leaves. Gas exchange and D13C measurements were determined at both high light (HL; 2000 lmol quanta m2 s1) and low light (LL;
150 lmol quanta m2 s1). Leaf material for d13C was collected from a similar aged leaf not used for gas exchange. Measurements were made on
3–5 leaves from separate plants for each species and shown are the means 6SE. Different letters indicate significant differences (P < 0.05) between
subtypes and measurement conditions.
D13C&
d13C&
Net CO2 assimilation
rate (lmol m2 s1)
pi/pa
HL
LL
HL
LL
HL
LL
Subfamily: Panicoideae
Tribe: Andropogoneae (NADP-ME)
Sorghum bicolor
Themeda triandra
Zea mays
35.166.8
34.663.7
37.460.8
8.160.4
8.460.7
7.960.8
0.2260.03
0.3060.01
0.2060.01
0.2160.04
0.5060.04
0.3460.06
4.060.1
4.260.2
3.660.3
5.560.6
3.761.1
5.760.6
–12.260.3
–12.060.1
–12.460.1
Subfamily: Chloridoideae
Tribe: Main Chloroid assemblage (NAD-ME)
Astrebla lappacea
Astrebla pectinata
Eragrostis superba
45.464.5
43.962.0
43.660.8
7.660.3
Nd
7.460.1
0.2260.01
0.2660.04
0.2760.03
0.3160.04
Nd
0.4260.03
4.460.6
4.660.1
4.660.4
5.560.5
Nd
6.961.1
–13.360.3
–11.860.5
–12.960.7
Subfamily: Panicoideae
Tribe: Paniceae (NADP-ME)
Cenchrus ciliaris
Chrysopogon fallax
Paspalum dilatatum
Pennisetum clandestinum
44.863.7
40.362.6
33.761.4
39.662.4
8.360.8
8.060.7
7.460.1
8.160.6
0.2960.03
0.2960.01
0.3460.01
0.3160.01
0.1760.05
0.2860.02
0.4260.03
0.5160.07
3.560.2
4.960.1
3.960.9
3.560.6
7.462.3
5.560.6
6.961.1
4.560.6
–12.060.1
–12.360.2
–13.060.2
–11.760.1
Subfamily: Panicoideae
Tribe: Paniceae (NAD-ME)
Panicum coloratum
Panicum miliecium
Andropogoneae (NADP-ME)
Chloroideae (NAD-ME)
Paniceae (NADP-ME)
Paniceae (NAD-ME)
35.760.7
35.360.8
35.761.1a
44.361.0b
40.560.9c
36.561.1a
8.960.6
7.960.4
8.260.2a
7.560.2a
7.960.2a
8.460.7a
0.2860.05
0.2960.02
0.2460.04a
0.2560.02a
0.3160.01a
0.2960.01a
0.2560.01
0.36 + 0.03
0.3560.10a
0.3760.07a
0.3560.09a
0.3060.08a
3.960.3
3.760.2
3.960.2a
4.560.1a
4.060.4a
3.860.2a
4.761.5
7.260.6
4.960.8a
6.260.9a
6.160.8a
5.961.8a
–13.460.2
–13.260.4
–12.260.2a
–12.660.5ab
–12.260.3a
–13.360.1b
NADP-ME
NAD-ME
37.961.6a
40.862.4a
8.060.1a
7.960.4a
0.2860.02a
0.2760.01a
0.3560.05a
0.3460.04a
3.960.2a
4.260.2a
5.660.5a
6.160.7a
–12.260.1a
–13.060.1b
Plant type
of PEP carboxylation (Vp). Values of leakiness (/) were estimated
by rearranging Equation 1 and solving for /.
Calculations of C18OO isotope discrimination
Discrimination against C18OO (D18O) when H2O and CO2 at the
site of exchange are at full isotopic equilibrium (h¼1) can be
predicted as (Farquhar and Lloyd, 1993)
D18 O ¼
a# þ eDea
1 eDea
ð3Þ
where a# is the diffusional discrimination, calculated for each
species as described by Farquhar and Lloyd (1993), and e is
calculated as pi/(pa–pi). There was little variation in a’ as the
average value for all species was 7.960.1&. The 18O enrichment of
CO2 compared with the atmosphere at the site of exchange in full
oxygen isotope equilibrium with the water was calculated as
(Cernusak et al., 2004)
Dea ¼
de ð1 þ ew Þ þ ew da
1 da
ð4Þ
where the equilibrium fractionation between water and CO2 (ew)
was determined at the LI-6400 measured leaf temperature (;30 C)
as described by Cernusak et al. (2004) and Cousins et al. (2006b).
The d18O of H2O at the sites of evaporation within a leaf (de) can
be estimated from the Craig and Gordon model of evaporative
enrichment (Craig and Gordon, 1965; Farquhar and Lloyd, 1993)
de ¼ ds þ ek þ eþ þ ðda ds ek Þ
ea
ei
ð5Þ
where ea and ei are the vapour pressures in the atmosphere and
the leaf intercellular spaces. da and ds are the isotopic composition
of water vapour in the air and source water, and the water taken
up by the plant (ds ¼ –5.360.3&), respectively. The kinetic
fractionation during diffusion of H2O from leaf intercellular air
spaces to the atmosphere (ek) and the equilibrium fractionation
between liquid water and water vapour (e+) was calculated
according to Cernusak et al. (2004) and Cousins et al. (2006b).
During the online gas exchange measurements, the leaves
remained in steady-state conditions for a minimum of 1.5 h and
under those conditions the value of ds is equal to the isotopic
composition of water transpired by the leaf (dt) (Harwood et al.,
1998). The air flowing into the leaf chamber was dry but
well mixed with transpired H2O within the chamber such that
the average relative humidity within the leaf chamber was
39.460.9%.
The proportion of CO2 in isotopic equilibrium with water at the
site of oxygen exchange (h) can be estimated from
h¼
Dca þ a#=ðe þ 1Þ
Dea þ a#=ðe þ 1Þ
ð6Þ
where Dca is the oxygen isotope composition of CO2 at the site of
exchange during photosynthesis determined by
1698 Cousins et al.
Dca ¼
D18 O a#
1 þ D18 O e
ð7Þ
It has been suggested that the extent of h in a leaf can also be
calculated from in vitro CA assays coupled with the unidirectional
flux of CO2 into the leaf (Gillon and Yakir, 2000a, b, 2001) from
the equation initially developed by Mills and Urey (1940):
h ¼ 1 eððCAleaf =Fin Þ=3Þ
ð8Þ
where CAleaf/Fin represents the mean number of hydration reactions
for each CO2 molecule inside the leaf (ks) (Gillon and Yakir, 2001).
Leaf CA activity (CAleaf) is determined as the product of the CA
hydration rate constant (kCA, mol m2 s1 bar1) and the mesophyll
pCO2 (pm). The rate constant kCA is calculated from in vitro
measurements of CA activity in leaf extracts (see below). The gross
influx of CO2 into a leaf [Fin¼gt pa; where gt is the total
conductance of CO2 from the atmosphere to the site of CO2–H2O
oxygen exchange (Gillon and Yakir, 2000a)] as well as pm
determine the residence time (s¼pm/Fin) of CO2 within the leaf.
Enzyme activities
CA activity was determined on ;1 cm2 section taken from the
same leaves used for gas exchange. Leaf samples were collected
after the gas exchange measurements and subsequently frozen in
liquid nitrogen and stored at –80 C. Tissue was ground on ice in
600 ll of extraction buffer [50 mM HEPES-KOH, pH 7.4, 10 mM
dithiothreitol (DTT), 1% polyvinlypolypyrrolidone (PVPP), 1 mM
EDTA, 0.1% Triton] with 20 ll of protease inhibitor cocktail
(Sigma, St Louis, MO, USA) and briefly centrifuged. CA activity
was measured on leaf extracts using mass spectrometry to measure
the rates of 18O2 exchange from labelled 13C18O2 to H16
2 O (Badger
and Price, 1989; von Caemmerer et al., 2004). Measurements of
leaf extracts were made at 25 C with a subsaturating total carbon
concentration of 1 mM. The hydration rates were calculated from the
enhancement in the rate of 18O loss over the uncatalysed rate, and
the non-enzymatic first-order rate constant was applied (pH 7.4,
appropriate for the mesophyll cytosol). The CA activity was reported
as a first-order rate constant kCA (mol m2 s1 bar1), and kCApm
gives the in vivo CA activity at that particular cytosolic pCO2.
Dry matter d13C
A similar leaf to the one used during gas exchange was collected,
oven-dried at 70 C, and ground with a mill ball. A subsample
of ground tissue was weighed and the isotopic composition
determined by combustion in a Carlo Erba elemental analyser,
and the CO2 was analysed by mass spectrometry. The d was
calculated as [(Rsample–Rstandard)/Rstandard]31000, where Rsample
and Rstandard are the 13/12C of the sample and the standard V-Pee
Dee Belemnite, respectively.
Results
Net CO2 assimilation rates
When plant species were grouped into photosynthetic
subtypes (NAD-ME versus NADP-ME), the rates of net
CO2 assimilation were similar between the two groups at
both high (2000 lmol quanta m2 s1) and low
(150 lmol quanta m2 s1) measuring light conditions
(Table 1). However, if net CO2 assimilation was analysed
by species within a tribe and photosynthetic subtypes (see
Table 1) then the Chloroideae-NAD and the PaniceaeNADP had higher rates compared with the AndropogoneaeNADP and Paniceae-NAD under high light conditions
only (Table 1). The ratio of intercellular to ambient CO2
partial pressures (pi/pa) was similar between the NAD-ME
and NADP-ME subtypes, and there was no difference in
pi/pa between species of different tribes (Table 1).
Isotope discrimination
There was no difference in photosynthetic carbon isotope
discrimination (D13C) between photosynthetic subtypes,
nor was there any difference in D13C between tribes at
either light level (Table 1). In all species measured, except
for Themeda traiandra, D13C was higher at low light
compared with high light (Table 1). The carbon isotope
composition of leaf material (d13C) determined on
a similar aged leaf as used for the gas exchange measurements was more depleted in the heaver isotope (13C) in
the NAD-ME compared with the NADP-ME and more
depleted in the Paniceae-NAD species compared with
those species in the Andropogoneae-NADP and PaniceaeNADP tribes (Table 1).
The measured photosynthetic oxygen isotope discrimination (D18O), measured at high light only, was similar
between species in the NAD-ME and NADP-ME subtypes
as well as in the different tribes (Table 2). The leaf CA
content expressed as the rate constant (kCA, mol m2 s1
bar1) was also similar between all the NAD-ME and
NADP-ME subtypes (Table 2). When CA was expressed
as a rate of CO2 hydration (CAleaf), which takes into
account the internal CO2 concentration within the leaf, the
rates were similar between the NAD-ME and NADP-ME
subtypes but higher in the Paniceae compared with both
the Chloroideae and the Andropogoneae species (Table 2).
The ratio of the rate of PEPC carboxylation, estimated
from the rates of net CO2 assimilation, to the rate of CO2
hydration estimated from CA activity (Vp/Vh) was similar
in the NAD-ME and NADP-ME subtypes but lower in the
Paniceae compared with species in the other two tribes
(Table 3). The rate of CO2 leakage across the BSCs (/:
defined as the fraction of CO2 fixed by PEPC that
subsequently leaks out of the BSCs) was similar between
species within the two photosynthetic subtypes as well as
between species in different tribes (Table 3). The values
of / calculated with the estimates of Vp/Vh were lower in
all species than when Vp/Vh was not considered in the /
calculations (Table 3).
Isotope discrimination versus pi/pa and the extent of
isotopic equilibrium
The majority of the measured values of D13C fall within
the theoretical relationship of D13C to pi/pa as predicted
from the model of C4 carbon isotope discrimination
Isotope discrimination in C4 grasses 1699
Table 2. Oxygen isotope discrimination and leaf carbonic
anhydrase
List of NAD-ME and NADP-ME C4 species used in this study, online
oxygen isotope discrimination (D18O), the rate constant of leaf CA
(kCA), and leaf CA activity (CAleaf) measured in the youngest fully
expanded leaves. Measurements were made on 3–5 leaves from separate
plants for each species and shown are the means 6SE. Different letters
indicate significant differences (P < 0.05) between subtypes and
measurement conditions.
D18O
kCA
CAleaf
Subfamily: Panicoideae
Tribe: Andropogoneae (NADP-ME)
Sorghum bicolor
11.661.7
Themeda triandra
7.460.6
Zea mays
12.460.8
1.660.1
1.260.1
2.560.2
9866
146616
113633
Subfamily: Chloridoideae
Tribe: Main Chloroid
assemblage (NAD-ME)
Astrebla lappacea
Astrebla pectinata
Eragrostis superba
6.560.9
12.260.7
8.561.3
2.160.1
2.260.1
2.460.1
119611
175635
204623
Subfamily: Panicoideae
Tribe: Paniceae (NADP-ME)
Cenchrus ciliaris
Chrysopogon fallax
Paspalum dilatatum
Pennisetum clandestinum
9.360.5
10.161.4
7.660.9
9.861.2
3.160.3
2.060.1
2.060.2
2.560.4
300650
21366
272629
281628
Subfamily: Panicoideae
Tribe: Paniceae (NAD-ME)
Panicum coloratum
Panicum miliecium
6.560.5
11.062.3
3.160.2
2.460.1
236653
224658
Andropogoneae (NADP-ME)
Chloroideae (NAD-ME)
Paniceae (NADP-ME)
Paniceae (NAD-ME)
10.561.9a
9.062.1a
9.2 + 0.7a
8.861.3a
1.860.5a
2.260.1a
2.460.3a
2.760.5a
119617a
165630a
266622b
23069b
Plant type
NADP-ME
NAD-ME
9.860.8a 2.160.3a 203634a
9.061.3a 2.460.2a 191623a
developed by Farquhar (1983) (Fig. 1). The theoretical
relationship of D13C and pi/pa was calculated with a /
value of 0.25, and the initial CO2 carboxylation reaction
catalysed by PEPC relative to the CO2 hydration by CA
(Vp/Vh) was assumed to be either zero (solid line) or 0.4
(dashed line; Fig. 1). The 0.4 value for Vp/Vh was taken as
the maximum value estimated in Table 3. Values of D13C
in the Andropogoneae and the Chloroideae are generally
higher than in the Paniceae, but this trend was not
significantly different (Table 1; Fig. 1). Measured D18O
was higher in the C4 dicots compared with the C4
monocots as would be predicted from Equation 3 based
on the higher pi/pa values in the dicots (Fig. 2). However,
D18O showed little relationship with pi/pa within the
dicots and monocots, unlike the model predictions, and
D18O values were substantially lower at each pi/pa than
predicted from Equation 3 (Fig. 2). Additionally, the
proportion of CO2 in isotopic equilibrium with water at
the site of oxygen exchange (h) predicted from the
number of hydration reactions per CO2 molecule (ks) was
substantially higher than the h values determined from
D18O measurements in all species including the C4 dicots
Table 3. The ratio of Vp/Vh and leakiness
List of NAD-ME and NADP-ME C4 species used in this study, the ratio
of PEPC and CA activity (Vp/Vh), and bundle sheath leakiness estimated
without and with factoring CA activity. Measurements were made on
3–5 leaves from separate plants for each species and shown are the
means 6SE. Different letters indicate significant differences (P < 0.05)
between subtypes and measurement conditions.
Plant type
Vp/Vh
/
/ with CA
Subfamily: Panicoideae
Tribe: Andropogoneae
(NADP-ME)
Sorghum bicolor
Themeda triandra
Zea mays
0.3860.09
0.2560.06
0.2860.06
0.2960.01
0.3360.02
0.2260.06
0.1660.03
0.2460.04
0.1360.06
Subfamily: Chloridoideae
Tribe: Main Chloroid
assemblage (NAD-ME)
Astrebla lappacea
Astrebla pectinata
Eragrostis superba
0.4060.05
0.2460.04
0.2360.03
0.3760.10
0.3860.01
0.3860.06
0.2260.09
0.2960.01
0.2960.06
Subfamily: Panicoideae
Tribe: Paniceae (NADP-ME)
Cenchrus ciliaris
Chrysopogon fallax
Paspalum dilatatum
Pennisetum clandestinum
0.1860.01
0.1960.01
0.1560.02
0.1660.01
0.2560.03
0.4260.01
0.3160.10
0.2660.06
0.1860.03
0.3460.01
0.2560.09
0.2060.06
Subfamily: Panicoideae
Tribe: Paniceae (NAD-ME)
Panicum coloratum
Panicum miliecium
0.1460.03
0.1560.02
0.3060.04
0.2760.03
0.2460.04
0.2160.04
Andropogoneae (NADP-ME)
Chloroideae (NAD-ME)
Paniceae (NADP-ME)
Paniceae (NAD-ME)
0.3160.05a
0.2960.07a
0.1760.01b
0.1560.01b
0.2860.04a
0.3860.03a
0.3160.04a
0.2860.02a
0.1860.04a
0.2760.03a
0.2460.04a
0.2260.02a
NADP-ME
NAD-ME
0.2260.03a 0.3060.03a 0.2160.03a
0.2360.05a 0.3460.03a 0.2560.02a
which have higher ks values compared with the C4
monocots (Fig. 3). The value of h in the C3 dicot was close
to the theoretical value of h estimated from ks values.
Discussion
Leaf 13C isotope composition and photosynthetic
discrimination
As previously reported (Hattersley, 1982; Ohsugi et al.,
1988), the leaf material of species in the NAD-ME
subtype was significantly depleted in 13C compared with
the NADP-ME species (Table 1). The depletion in 13C in
the NAD-ME species has been attributed to difference in
leakiness between the two subtypes (Hattersley, 1982;
Farquhar, 1983; Ohsugi et al., 1988; Buchmann et al.,
1996), and it has been hypothesized that the presence of
a suberized lamella in the BSCs of the NADP-ME species
reduces the conductance of CO2 across the BSCs.
However, the present measurements of D13C under high
light conditions indicated that leakiness does not vary
between the subtypes and does not support the notion that
d13C differs between the subtypes because of differences
in leakiness (Tables 1, 3; Fig. 1). This is similar to
1700 Cousins et al.
Fig. 1. Carbon isotope discrimination (D13C) as a function of the ratio
of intercellular to ambient pCO2 (pi/pa). The lines represent the
theoretical relationship of D13C and pi/pa where /¼0.25, and the ratio
of the PEPC carboxylation to the CO2 hydration reaction (Vp/Vh) is
either 0 (solid line) or 0.4 (dashed line); see Equation 1. Each point
represents the means 6SE of measurements made on 3–5 leaves from
separate plants for each species. Symbols represent the NADP-ME
monocots (filled diamonds and stars), NAD-ME monocots (open circles
and stars), NADP-ME C4 dicots (filled triangle), and NAD-ME dicot
(open triangle). Data for the dicot C4 NAD-ME and NADP-ME are
adapted from Cousins et al. (2006a, 2007).
Fig. 2. Oxygen isotope discrimination (D18O) as a function of the ratio
of mesophyll cytosolic to ambient CO2 partial pressure (pi/pa). The line
represents the theoretical relationship of D18O and pi/pa at full isotopic
equilibrium where a# was ;7.960.1& and Dea¼33.7& (Equation 3),
and the CO2 supplied to the leaf had a d18O of 24& relative to
VSMOW. Each point represents the means 6SE of measurements made
on 3–5 leaves from separate plants for each species, and symbols are as
in Fig. 1. Data for the dicot C4 NAD-ME and NADP-ME are adapted
from Cousins et al. (2006b, 2007).
Fig. 3. The extent of isotopic equilibrium (h) as a function of the
number of hydration reactions per CO2 molecule (ks). Measured values
of h were determined from D18O using Equation 6. Each point
represents a measurement made on an individual leaf for a single
species, and symbols are as in Fig. 1 and a C3 dicot (asterisk). Data for
the C3 dicot and C4 NAD-ME and NADP-ME dicots are adapted from
Cousins et al. (2006a, 2007). The line represents the theoretical
relationship between h and ks (Equation 8).
previous estimates of leakiness in the NAD-ME and
NADP-ME species with various techniques (Krall and
Edwards, 1990; Hatch et al., 1995) including online
measurements of D13C (Henderson et al., 1992). The
quantum yield of CO2 assimilation has been reported as
a good proxy for estimating leakiness in C4 plants and that
the range of quantum yields measured in numerous C4
species reflects the differences in leakiness in these
species (Ehleringer and Pearcy, 1983; Farquhar, 1983).
However, the greatest difference in the quantum yield is
generally not seen between NAD-ME and NADP-ME
subtypes but between monocots and dicots (Ehleringer
and Pearcy, 1983; von Caemmerer and Furbank, 2003).
Additionally, it is difficult to estimate leakiness using the
quantum yield measurement given the uncertainties in the
production of ATP from absorbed quanta and how this
ATP/quanta ratio varies between species (Furbank et al.,
1990; von Caemmerer and Furbank, 2003).
The quantum yield of CO2 fixation is typically determined from the initial slope of a light response curve,
and low light growth conditions have previously been
shown to decrease the 13C isotopic signature of C4 leaf
material (Buchmann et al., 1996; Kubásek et al., 2007).
Additionally, reducing the light level during online
measurements of D13C causes an increase in the C4
photosynthetic isotopic fractionation against 13CO2
(Henderson et al., 1992; Cousins et al., 2006a; Tazoe
et al., 2006). It is possible that low light conditions alter
the co-ordination between the C4 and C3, causing the
Isotope discrimination in C4 grasses 1701
overcycling of CO2 and increasing leakiness; however,
a mechanism has not yet been reported. D13C was
measured under low light conditions (150 lmol quanta
m2 s1) to investigate whether or not D13C responded
differently in the two C4 subtypes (Table 1). As previously reported, D13C increased at low light (Henderson
et al., 1992; Cousins et al., 2006a; Tazoe et al., 2006), but
there was no significant difference in the response of D13C
in the C4 subtypes under low light. This indicates that the
low light enhancement of D13C is widespread in C4 plants
but does not provide an explanation for the differences in
d13C between the subtypes. It is interesting to note that the
method of determining the quantum yield of CO2 assimilation from the initial slope of the photosynthetic light
response curve may be difficult to interrupt as low light
appears to alter the co-ordination of the C4 and C3 cycles.
Photosynthetic discrimination does not readily explain
the time-tested differences in d13C between the NAD-ME
and NADP-ME C4 subtypes, which suggests that differences in post-photosynthetic discrimination of photoassimilate may be responsible for the d13C differences.
As suggested over a decade ago, further studies should be
conducted to investigate the differences in respiratory
discrimination in a variety of NAD-ME and NADPME C4 species (Henderson et al., 1992).
C18OO discrimination
There was no difference in the photosynthetic C18OO
discrimination (D18O) between the C4 species measured in
this study (Table 2). Values of D18O showed little increase
with an increase in pi/pa, and the measured D18O was
significantly less in all species than would be predicted
from the theoretical relationship of D18O and pi/pa
(Equation 3; Fig. 2). The low D18O in the C4 grasses is
similar to what was measured in the C4 dicots A. edulis
and F. bidentis (Fig. 2), indicating that D18O is low in C4
plants regardless of their evolutionary origin, and low
D18O may indeed be inherent to C4 photosynthesis (Gillon
and Yakir, 2000b, 2001) but not necessarily related to low
CA activity (see below).
Carbonic anhydrase
There was no significant difference in the CA content,
presented as the rate constant (kCA), nor leaf activity
(CAleaf) between the NAD-ME and NADP-ME species
(Table 2). The values of CAleaf for the C4 grasses are
significantly less then previously reported for C4 dicots
including Amaranthus sp., 3896119 lmol m2 s1 (Gillon
and Yakir, 2001); A. edulis, 7836153 lmol m2 s1
(Cousins et al., 2007); and F. bidentis, 13216114 lmol
m2 s1 (Cousins et al., 2006a, b). Our data support the
suggestion that low CA may not be due to or explicitly
associated with C4 photosynthesis, but that it may instead be
a trait of the clade of grasses to which the species in this
current study belong (Edwards et al., 2007). Within the C4
grasses, CA activity is not consistent as the species within
the Andropogoneae and Chloroideae had ;60% of the
CAleaf activity compared with species of the Paniceae (Table
2). The ratio of the initial photosynthetic carboxylation
reaction catalysed by PEPC to the rate of CO2 hydration by
CA (Vp/Vh) averaged 0.2460.03 in all the grasses and was
significantly lower in the Paniceae species compared with
the other species (Table 3). Values of Vp/Vh reported here
are similar to values reported in an earlier publication
which suggested that CA activity may be just sufficient
to support rates of photosynthesis in C4 plants (Hatch
and Burnell, 1990). As noted above, there were no
differences in D13C or D18O between the photosynthetic
subtypes and the tribes, indicating that differences in CA
activity do not have a large influence on the photosynthetic isotope exchange between the C4 grass species.
However, Vp/Vh is low enough to have a major influence
on D13C in all the C4 grasses measured here (Fig. 1;
Table 3). For example, variation in D13C not explained
by pi/pa (Fig. 1) can be explained from estimates of
Vp/Vh. Additionally, taking Vp/Vh into consideration
reduced the estimated values of leakiness by ;20%
(Table 3). This has implications for interpreting the
contribution of C4 photosynthesis to regional CO2
exchange in C4-dominated grasslands as the photosynthetic discrimination against 13CO2 may be underestimated if CA activity is not taken into consideration.
CO2 and H2O isotopic equilibrium
The exchange of 18O between CO2 and H2O is facilitated
by CA, which catalyses the interconversion of CO2 and
bicarbonate (HCO–3), and high CA activity will increase
the proportion of CO2 in isotopic equilibrium with the
H2O (h). The extent of h measured from D18O (Equation
6) was low in all species compared with h estimated from
the number of hydration reactions per CO2 (ks) (Equation
8; Fig. 3). This suggests that the total leaf CA activity in
C4 monocots does not represent the CA activity associated
with the CO2–H2O oxygen exchange which influences
D18O. This finding is similar to what was previously
reported for C4 dicots which have 2.5 times the CA
activity and values of ks compared with the C4 grasses
(Cousins et al., 2006b, 2007) (Fig. 3).
The present study suggests that in C4 species leaf CA
activity cannot readily be used as an indicator of the
extent of 18O equilibration. This might be because not all
of the CA located in mesophyll cytosol in C4 species is
available at the site of CO2–H2O exchange compared with
C3 species where CA is located in chloroplasts which
appress intercellular airspaces (Poincelot, 1972; Ku and
Edwards, 1975). Alternatively, the extent of 18O equilibration may be miscalculated if the estimated values of de
do not accurately describe the isotopic composition of
H2O at the site of oxygen exchange between leaf H2O and
CO2 (Cousins et al., 2006b).
1702 Cousins et al.
Conclusions
Differences in the carbon isotope composition (d13C)
between the NAD-ME- and NADP-ME-type C4 grasses
are not explained by photosynthetic carbon isotope
discrimination (D13C) at either high or low light. This
indicates that other processes such as post-photosynthetic
discrimination have a major influence on d13C in C4
grasses. The low CA in the C4 grasses may influence
D13C, and CA activity should be considered when
estimating CO2 bundle sheath leakiness and the contribution of C4 photosynthesis to the isotopic signature of
atmospheric CO2 in C4-dominated ecosystems. Additionally, leaf CA activity does not appear readily to predict the
extent of 18O equilibration between leaf H2O and CO2.
Acknowledgements
We thank Sue Wood for the carbon isotope analysis of dry matter
samples, and Oula Ghannoum for supplying the seeds used in this
study. ABC was supported in part by an NSF international
postdoctoral fellowship.
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