Plant Physiol. (1980) 66, 931-934
0032-0889/80/66/093 1/04/$00.50/0
Diffusional Contribution to Carbon Isotope Fractionation during
Dark CO2 Fixation in CAM Plants'
Received for publication March 21, 1980 and in revised form June 25, 1980
MARION H. O'LEARY AND C. BARRY OSMOND
Departments of Chemistry and Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 and
Department of Environmental Biology, Research School of Biological Sciences, Australian National University,
Canberra 2601, Australia
requires measurement of the isotopic composition of the appropriate carbon atom in an early product of carbon fixation. In the
A mathematical model is developed which can be used to predict in vivo
case
of C4 photosynthesis, this would logically be carbon-4 of
carbon isotope fractionations associated with carbon fixation in plants in
terms of diffusion, CO2 hydration, and carboxylation components. This photosynthetically formed oxalacetate, malate, or aspartate.
Plants exhibiting CAM show variable isotope discriminations,
model also permits calculation of internal CO2 concentration for comparison with results of gas-exchange experments. The isotope fractionations reflecting the contributions of the CO2 fixed in the dark by PEP
associated with carbon fixation in Kaawchoi daigremontiana and Bryophyl- carboxylase and in the light by RuP2 carboxylase (1, 22-24). CAM
bun tubiflorm have been measured by isolation of malc acid following plants are particularly useful for investigating components of
dark fixation and enzymic determination of the isotopic composition of carbon isotope fractionation because during dark CO2 fixation in
carbon4 of this material. Corrections are made for residual malc acid, these plants a large pool of malic acid accumulates as a result of
fumarase activity, and respiration. Comparison of these data with calcula- PEP carboxylase activity (22) and is subject to little further
tions from the model indicates that the rate of carbon fixation is limited metabolism until the following light period. Here, isolation of this
principally by diffusion, rather than by carboxylation. Processes subsequent malic acid and measurement of the isotopic composition of carto the initial carboxylation also contribute to the over-all isotopic compo- bon-4 are reported, along with a model which can be used to
sition of the plant.
explain this isotopic composition in terms of known in vitro
fractionations associated with diffusion, CO2 hydration, and carABSTRACT
boxylation.
MATERIALS AND METHODS
Plants were grown in a well-vented growth chamber with a 10h light period at 23 C and a 14-h dark period at 17 C. Fully
expanded leaves of mature plants were harvested at the end of the
dark period and at the end of the light period. A sample was saved
for analysis of whole leaf 8 13C by combustion. The remaining
material (5-10 g) was homogenized in 80%1o methanol, boiled for
5 min, cooled, and centrifuged, and the solution was evaporated
to dryness on a rotary evaporator. The redissolved residue was
filtered and then chromatographed on a 1.2- x 15-cm column of
Dowex 50-H+ eluted with H20. Fractions containing malic acid
[identified by enzymic analysis with malic enzyme (11)] were
combined and chromatographed on a 1.2- x 40-cm column of
Dowex 1 formate form eluted with a linear gradient (600 ml) of 0
to 6 M formic acid. Malic acid-containing fractions were combined
and evaporated to dryness, particular care being taken to remove
last traces of formic acid. The melting point of these malic acid
samples was 92-101 C [Lit. 98-99 C (8)]. Control samples of malic
acid could be carried through this procedure with no change in
isotopic composition.
Values of 8 13C for the whole leaf and for malic acid were
determined by combustion. For determination of 8 13C of carbon4 of malic acid, a solution containing 0.1 mmol malic acid, 0.2
mmol oxidized glutathione, 1 ,umol NADP, and 20 ,umol Mn2+ in
10 ml 0.1 M Hepes buffer (pH 7.5) was freed of dissolved CO2 by
purging with CO2-free N2 for 4 h (21). Malic enzyme and glutathione reductase (Sigma Chemical Co.) were freed of dissolved
CO2 by anaerobic gel filtration (21). Sufficient malic enzyme and
glutathione reductase were added to the degassed malate solution
Plants discriminate against 13C during CO2 fixation. It is generally assumed that this discrimination is due to isotope fractionation by the pertinent carboxylase (2, 15, 32). Experiments conducted with isolated carboxylases are qualitatively consistent with
this assumption, in that both C3 plants and the associated RuP22
carboxylase show comparatively large isotope discriminations (3,
9, 31), whereas C4 plants and PEP carboxylase show small discriminations (28, 31, 34).
However, this assumption of equivalency may be incorrect for
two reasons. First, carbon may be fractionated during CO2 loss
associated with respiration or refixation. Second, events other than
carboxylation itself might affect the initial isotope fractionation.
Stomatal diffusion, for example, might contribute either because
diffusion fractionates isotopes (16) or because diffusion reduces
the extent to which the carboxylase isotope fractionation is expressed.
Because of possible contributions from postcarboxylation
events, isotopic composition data for whole plants are inadequate
for quantitative testing of the assumed equivalency between in
vivo and in vitro isotope fractionations. Instead, in vivo testing
' This work was sponsored by National Science Foundation Grant
PCM77-00812, by National Science Foundation Grant INT 78-21644
through the United States-Australia cooperative program, and by a visiting
fellowship (to M. H. 0.) at the Australian National University.
2Abbreviations: RuP2, ribulose bisphosphate; PEP, phosphoenolpyruvate; CAM, Crassulacean acid metabolism.
931
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1980 American Society of Plant Biologists. All rights reserved.
O'LEARY AND OSMOND
932
that decarboxylation should be complete in 2 h, but the reactions
were allowed to proceed for at least 12 h. Less than 0.1% of the
initial malate remained at the end of the decarboxylation. After
12 h, the decarboxylation solution was acidified with 2 ml 10 M
H2SO4 and the CO2 produced was isolated and freed of H20 on
a vacuum line.
Measurements of 8 '3C were made on a Micromass 602 mass
spectrometer. Measurements of multiple samples showed that the
reproducibility of the entire procedure was generally ±0.5%o. The
8 3C value for growth chamber CO2 was -7 ± l%o.
For gas exchange, leaves were enclosed in the open-system gasexchange system described previously (27) and CO2 uptake was
monitored continuously throughout the dark period. The rate of
CO2 uptake was calculated at frequent intervals throughout the
dark period and these measurements were used to calibrate the
continuous record.Total CO2 uptake was calculated from the area
under the curve showing the nocturnal CO2 uptake pattern. Malic
acid content of the opposite, near identical leaf was measured
before the dark period began and that of the leaf in the chamber
was measured at the end of the dark period. Malic acid content
was measured by titration and by enzymic analysis (10, 24). Under
these conditions the malic acid accounted for 93% of the total CO2
fixed in the dark (Table I).
THEORY
Isotopic compositions are measured as an abundance ratio (R)
(6):
'3CO2/'2C02
or as a 8 value, in units per mil (%o):
R
(1)
=
8 (%o) = [Ra
_ I]
x
1000
(2)
The standard is PDB (Belemnite from the Pee Dee Formation in
South Carolina) (6, 15). Organic matter is invariably depleted in
13C compared to PDB, so 8 values of organic materials are
negative. A less negative figure means richer in 13C, or "heavier."
Isotope discrimination is expressed as a difference in 8 value
between source and product (6, 15):
discrimination
=
8(source
3product
+ urce. 1000
-
l1
(3)
Plant Physiol. Vol. 66, 1980
instead in terms of rate constants (in essence, the reciprocals of
resistivities) for the various transformations involved because the
resulting differential equations made it possible to consider return
of materials from internal pools to the external pool (e.g. the
return of intercellular CO2 to the atmospheric CO2 pool). Quantitative treatment of carbon isotope fractionations requires inclusion of this return. The model here corrects a number of errors in
previous models (2, 15, 25) and is analogous to the treatment of
carbon isotope fractionations in multistep enzyme-catalyzed reactions (20).
The steps in the initial carbon fixation are summarized as
C02, ext
k2
ki fast
C02, i- HCO3 cell
k3 1 PEP
malate
+-
(6)
oxalacetate
Rate constant ki describes the diffusion of external CO2 into
the intercellular air space. It thus may include any external
boundary layer diffusion, stomatal diffusion, and perhaps an
internal diffusion component. Of these, the stomatal resistance to
diffusion is generally the most important. This diffusion will show
a carbon isotope fractionation of +4.4%o (16). Rate constant k2
reflects outward diffusion of C02, i and thus should also show a
carbon isotope fractionation of +4.4%o.
The next step represents absorption of CO2 at the air-liquid
interface, transport of CO2 into the cell, and equilibration of CO2
with HCO3 . It is assumed that absorption and transport are rapid.
The level of carbonic anhydrase is presumably adequate to maintain the C02-HC03- interchange at isotopic equilibrium (26). The
isotope fractionation associated with dissolution and hydration of
CO2 is-8%o at 25 C (17).
The next step is reaction of HCO3 with PEP. A carbon isotope
fractionation of +2%o is associated with this step if HCO3- is
considered to be the substrate (M. H. O'Leary, J. Slater, and J.
Rife, unpublished data; cf refs. 28 and 34). When modelling in
vivo isotope fractionation it is useful to combine the absorption,
liquid-phase transport, CO2 hydration, and carboxylation steps.
When this is done, the net fractionation for PEP carboxylase in
vivo from C02, i to oxalacetate is -6%o. This factor has frequently
been misinterpreted (28, 32). No isotope fractionation should be
associated with the reduction of oxalacetate to malate (20).
Under conditions of steady-state photosynthesis, the rate of
change of CO2, i with time should be essentially zero. With this
assumption, the relationship between the isotopic composition of
atmospheric CO2 (Rt.) and that of carbon4 of malate (Rma.) can
be derived. Note that these are isotope ratios, rather than 8 13C
values:
effect," k 2/k 3, the ratio of rate constants for
reactions of the respective isotopic substances. When the quantity
of source is large compared to the amount of product
k 12/k 13 = Rsource/Rproduct
(4)
Thus, the discrimination factor is given by
discrimination = 1000 x [1- (k13/k'2)]
(5) i
Most models for photosynthetic carbon fixation have been
Ratm El E3/E2 + k3/k2
(7)
based on the "resistivities" associated with the various physical
1 + k3/k2
Rmai
and chemical steps in the process (12, 19). Here, work was done in which E1 =
k112/kil3, the isotope fractionation associated with
etc.
As noted above, these fractionations are known: E1 = E2
kl,
Table L. Measured Net CO2 Uptake and Malic Acid Synthesis in Leaves
= 1.0044; E3 = 0.994.
of K daigremontiana during 14-h Dark at 16 C
Thus, knowledge of the isotope fractionation into carbon-4 of
A Malic Acid
Experiment
CO2 uptake
malate makes it possible to calculate k3/k2. Qualitatively, there
are two limiting models which might explain the isotope fraction,nol
ation
produced in vivo via PEP carboxylase. The first model is the
1
1236
1155a; 1168b
commonly
accepted one, which assumes that carboxylation en2
1055
985a
limits
the rate of carbon fixation; that is, k3/k2 is small and
tirely
a Malic acid measured by titration.
the intracellular CO2 pool is in equilibrium with the atmosphere.
b Malic acid measured by enzymic assay.
Diffusional fractionation does not enter. Under these conditions,
or as an "isotope
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1980 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 66, 1980
ISOTOPE FRACTIONATION IN CAM
933
and Garnier-Dardart (7). This difference probably arises in part
from contributions of other acids to the Deleens samples and in
The second limiting model is one in which the rate is entirely part from differences in the isotopic compositions of the starch
controlled by diffusion, that is, k3/k2 is large and all internal CO2 pools (which provide carbons -1 to -3 of malate) in the two
taken up by carboxylation. Under these conditions, the isotopic experiments. Malate of similar isotopic composition to the malate
composition of carbon4 of malate should be near -1I %o.
used here has been isolated from a C4 plant (33). The isotopic
There is currently a considerable interest in the variation of composition of crystalline oxalate in several species of cactus has
stomatal resistance in CAM plants in response to CO2 concentra- recently been shown to be near -8%o (29).
tions (35, 36). Gas-exchange measurements and direct measureThe carbon isotope fractionation associated with CO2 fixation
ments of CO2j by GC have provided useful information (5). by the C4 pathway in CAM plants can be obtained by comparison
Carbon isotope fractionations provide another approach. For the of the isotopic composition of carbon-4 of malate with that of
model given in equation 6, it is expected that ki = k2, that is, the atmospheric CO2, provided that corrections to the former value
rate constant for CO2 diffusion is the same in both directions. The are made for (a) malate remaining at the end of the previous light
steady-state assumption with regard to CO2,i makes it possible to period; (b) any randomization due to furmarase; (c) contribution
of respiratory carbon. The fractionation then can be used in
show that
connection
with equations 7 and 8 to determine the relative
C02 e = 1 + (k3/k2)
(8) contributions of diffusion and carboxylation to the over-all CO2
C02,
incorporation rate. The corrections for K daigremontiana are
The ratio k3/k2 is, of course, obtained from the isotope fractiona- summarized in the following paragraphs.
The malate pool at the end of the light period is about 15% as
tion measurements, so a direct correlation is expected between gas
large as at the end of the dark period, and the 8 13C value for
exchange and isotope fractionation.
carbon-4 of this material is -6.4%o. This gives a correction of
-0.3%o to the 8 13C value for carbon-4 of malate at the end of the
RESULTS
dark period.
Correction can be made for randomization due to fumarase by
Values of 8 13C have been measured for whole leaf, for purified
assuming
that carbons-i, -2, and -3 of malate (which arise from
of
malic acid, and for carbon4
malic acid isolated from Kalanchoe
daigremontiana and from Bryophyllum tubiflorum at the end of the starch) all have the same isotopic composition (cf. Table II) and
dark period and at the end of the light period (Table II). Also that, as is frequently observed (4, 14) randomization of carbons- I
included are calculated isotopic compositions for carbons I to 3 of and -4 is about two-thirds complete. Thus, the correction to
carbon-4 of malate is +0.5%o.
malate.
The correction for respired carbon is the most difficult to make
Gas-exchange measurements on leaves of K. daigremontiana
comparable to those used for isotope fractionation measurements because neither the quantity of respired carbon nor its isotopic
showed that net CO2 uptake in the dark period was essentially composition is known. Respired carbon becomes part of the C02,
equivalent to the quantity of malic acid synthesized. From the pool (cf. equation 6) and should be subject to the same partitioning
data presented here and previous data (13) it is estimated that as CO2,J. If it is assumed that 10%o of the malate synthesized arises
about 10%o of the malate synthesized arises from respired carbon. from respired carbon and that respired carbon has the same 8 13C
value as whole leaf carbon, then the correction is +0.5%o.
The aggregate of the three corrections is, thus, +0.7%o, with
DISCUSSION
an uncertainty of about ±0.5%o, and the corrected value for 8 '3C
When CAM plants are allowed to assimilate CO2 only in fhe of carbon-4 of malate immediately following carboxylation is
dark, they show 8 13C values near -1 %o (18). The fact that 8 13C -7.4%o. Equation 7 makes it possible to calculate that k3/k2 = 1.6.
values for whole leaves of K. daigremontiana in the study here are That is, molecules in the CO2,J pool are taken up by carboxylation
1.6 times as often as they diffuse back to the atmosphere.
near -15%o indicates that some C3 carboxylation is occurring.
Thus, diffusion provides a significant barrier to carbon fixation
However, carbon4 of malic acid (after appropriate corrections)
arises from atmospheric CO2 and, thus, reflects any isotope frac- in CAM plants. It can be calculated that, if the diffusion barrier
tionation in the initial carbon fixation process, independent of any were removed, the rate of carbon fixation would increase by about
a factor of 3 but, of course, at considerable expense in terms of
C3 fixation.
The isotopic composition of malic acid in the K. daigremontiana water loss. On the other hand, if the carboxylation capacity of the
experiments (Table II) is about 2%o more positive than the corre- plant were substantially increased, the carbon fixation rate would
sponding values for the "acid fraction" in the work of Deleens increase by less than 50Yo. This partitioning of the CO2, pool
the isotopic composition of carbon4 of malate should be near
-1%o.
Table II. Carbon-13 Isotopic Compositions of Various Materials Isolatedfrom K daigremontiana and B.
tub!florum
Values given are the average of four determinations for K. daigremontiana, and the average of two for B.
tub7florum. Errors given are SD.
8 13C Relative to PDB
Malate Yield
Sample
Whole Leaf
Total Malate C4 of Malate
C1-C3 Malatea
jimoligfresh wt
%o
K daigremontiana
Morning
Evening
90 ± 10
15 ± 3
-13.9 ± 2.2
-13.7 ± 1.2
B. tubiflorum
50
-12.4
Morning
5
-13.1
Evening
a Calculated from % 8 13C (Cl - C3) + y4 8 13C (C4)
=
-9.1 ± 0.4
-9.2 + 0.5
-8.1 ± 0.7
-6.4 + 0.2
-9.4 ± 0.8
-10.1 + 0.6
-7.4
-5.3
-7.1
-4.1
8 13C (total malate).
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1980 American Society of Plant Biologists. All rights reserved.
-8.4
-8.1
934
O'LEARY AlN:D OSMOND
- w
probably represents the optimum balance between carboxylation
capacity, CO2 availability, and water use.
From this partitioning and equation 8 it can be calculated that
the average value of CO2,i during dark CO2 fixation is 130 ,ul/l.
Gas-exchange measurements (J.A.M. Holtum and S. C. Wong,
unpublished data) have been used to determine values of CO2J
under the conditions of these experiments. As in previous studies
(13, 15), values of CO2, change during the dark period. Early in
the dark period, when little carbon fixation is occurring, C02,
reaches a minimum value of 150 fl/l. Over the next several hours,
CO2,i increases more or less linearly, reaching a maximum of
about 250 ,tll just before sunrise.
The second aspect of the isotope fractionations which is of
interest is the comparison of the isotopic composition of carbon4
of malate with that expected for the whole plant under conditions
of totally CAM photosynthesis. There is a further isotope fractionation of about 4%o subsequent to the formation of malate. It
is possible that, during deacidification, there might be a small
leakage of CO2 to the atmosphere (10-20o), thus allowing a small
portion of the RuP2 carboxylase fractionation to be expressed.
CO2,i becomes quite high during deacidification (5), and there is
at least one report (28) that the CO2 evolved during deacidification
in K daigremontiana has a relatively positive 8 "3C value, as would
be required if this explanation is correct. The other possibility is
that this further fractionation is connected with respiratory processes.
The isotopic data for B. tubiflorum can be analyzed in a similar
way, but the uncertainties in this analysis are larger than in the
previous case. Corrections for fumarase and for respired carbon
are each near +1%o. The latter correction is subject to a considerable uncertainty. It is estimated that, after correction, the 8 "C
value for carbon-4 of malate is near -3.2%o, corresponding to k3/
k2= 0.27 and CO2, = 260 ,tl/l. Although these values must be
considered approximate, it is clear that diffusion is less limiting
than in the case of K daigremontiana.
The analysis based on equations 7 and 8 can be applied to all
types of plants, provided that the appropriate isotope fractionation
factors are available. Diffusional fractionations E1 and E2 will
always equal 1.0044. For C4 plants, E3 is 0.994. For C3 plants, the
value of E3 is somewhat uncertain, but it is probably near 1.03 (3,
9, 31). If postcarboxylation events are assumed not to contribute
significantly to the total isotope composition of the plant, then this
analysis suggests that the rate of diffusion plays an important role
in both C3 and C4 plants. The potential importance of diffusion in
C4 carboxylation has been noted by Schmidt and Winkler (30).
Acknowledgments-Peter Firth made the isotope ratio measurements in connection
with the whole plant studies. Johnathan Slater and James Rife measured the isotope
effect for purified PEP carboxylase. Professor R. Mills and Dr. K. Harris provided
valuable advice concerning diffusional isotope fractionation and Graham Farquhar
suggested the relationship to intercellular CO2 concentration.
LITERATURE CITED
1. BENDER MM, I ROUHANI, HM VINES, CC BLACK 1973 '3C/1 C ratio changes in
crassulacean acid metabolism plants. Plant Physiol 52: 427-430
2. BENEDICT CR 1978 Nature of obligate photoautotrophy. Annu Rev Plant Physiol
29: 67-93
3 CHRISTELLER JT, WA LAING, JR TROUGHTON 1976 Isotope discrimination by
ribulose-1,5-diphosphate carboxylase. No effect of temperature or HC03
concentration. Plant Physiol 57: 580-582
4. COCKBURN W, A MCAULAY 1975 The pathway of carbon dioxide fixation in
crassulacean plants. Plant Physiol 55: 87-89
5. COCKBURN W, IP TING, LO STERNBERG 1979 Relationships between stomatal
behavior and internal carbon dioxide concentration in crassulacean acid metabolism plants. Plant Physiol 63: 1029-1032
6. CRAIG H 1957 Isotopic standards for carbon and oxygen and correction factors
for mass spectrometric analysis of carbon dioxide. Geochim Cosmochim Acta
Plant Physiol. Vol. 66, 1980
12: 133-149
7. DELEENS E, J GARNIER-DARDART 1977 Carbon isotope composition of biochemical fractions isolated from leaves of Bryophyllum daigremontianum Berger, a
plant with crassulacean acid metabolism: some physiological aspects related to
CO2 dark fixation. Planta 135: 241-248
8. DUNSTAN AE, FB THOLE 1908 The relation between viscosity and chemical
constitution. II. The existence of racemic compounds in the liquid state. J
Chem Soc 93: 1815-1821
9. ESTEP MF, FR TABITA, PL PARKER, C VAN BAALEN 1978 Carbon isotope
fractionation by ribulose- 1,5-bisphosphate carboxylase from various organisms.
Plant Physiol 61: 680-687
10. HOHORST JH 1970 L(-)-Malat, Bestimmung mit Malat Dehydrogenase und
NAD. In HU Bergmeyer, ed, Methoden der enzymatischen Analyse. Verlag
Chemie, Weinheim, pp 1585-1589
11. Hsu RY, HA LARDY 1969 Malic enzyme. Methods Enzymol 13: 230-235
12. JARVIS PG 1971 The estimation of resistances to carbon dioxide transfer. In Z
Sestak, J Catsky, PG Jarvis, eds, Plant Photosynthetic Production Manual of
Methods, W. Junk, The Hague, pp 566-631
13. KAPLAN A, J GALE, A POIUAKOFF-MAYBER 1977 Effect of oxygen and carbon
dioxide concentrations on gross dark CO2 fixation and dark respiration in
Bryophyllum daigremontianum. Aust J Plant Physiol 4: 745-752
14. KLUGE M, L BLEY, R SCHMID 1975 Malate synthesis in crassulacean acid
metabolism (CAM) via a double CO2 dark fixation? In R Marcelle, ed,
Environmental and Biological Control of Photosynthesis. W. Junk, The Hague,
pp 28 1-288
15. LERMAN JC 1975 How to interpret variations in the carbon isotope ratio of plants:
biologic and environmental effects. In R Marcelle, ed, Environmental and
Biological Control of Photosynthesis. W. Junk, The Hague, pp 323-335
16. MASON EA, TR MARRERO 1970 The diffusion of atoms and molecules. Adv At
Mol Phys 6: 155-232
17. MOOK WG, JC BOMMERSON, WH STAVERMAN 1974 Carbon isotope fractionation
between dissolved bicarbonate and gaseous carbon dioxide. Earth Plan Sci Lett
22: 169-175
18. NALBORCZYK E, LJ LACROIX, RD HILL 1975 Environmental influences on light
and dark C02 fixation by Kalanchoe daigremontiana. Can J Bot 53: 1132-1138
19. NOBEL PS 1974 Biophysical Plant Physiology. WH Freeman, San Francisco, pp
325-335
20. O'LEARY MH 1978 Heavy-atom isotope effects in enzyme-catalyzed reactions. In
RD Gandour, RL Schowen, eds, Transition States of Biochemical Processes.
Plenum Publishing Corp, New York, pp 285-316
21. O'LEARY MH 1980 Determination of heavy-atom isotope effects on enzymecatalyzed reactions. Methods Enzymol 64B: 83-104
22. OSMOND CB 1978 Crassulacean acid metabolism: a curiosity in context. Annu
Rev Plant Physiol 29: 379-414
23. OSMOND CB, WG ALLAWAY, BG SUTTON, JH TROUGHTON, 0 QUEIROZ, U
LUTrGE, K WINTER 1973 Carbon isotope discrimination in photosynthesis of
CAM plants. Nature 246: 41-42
24. OSMOND CB, MM BENDER, RH BURRIS 1976 Pathways of CO2 fixation in the
CAM plant Kalanchoe daigremontiana. III. Correlation with 8 "3C value during
growth and water stress. Aust J Plant Physiol 3: 787-799
25. PARK R, S EPSTEIN 1960 Carbon isotope fractionation during photosynthesis.
Geochim Cosmochim Acta 21: 110-126
26. POINCELOT RP 1979 Carbonic anhydrase. In M Gibbs, E Latzko, eds, Encyclopedia of Plant Physiology, New Series Vol 6, Springer-Verlag, Heidelberg, pp
230-238
27. POWLES SB, CB OSMOND 1978 Inhibition of the capacity and efficiency of
photosynthesis in bean leaflets illuminated in CO2 free atmosphere at low
oxygen: a possible role for photorespiration. Aust J Plant Physiol 5: 619-629
28. REIBACH PH, CR BENEDICT 1977 Fractionation of stable carbon isotopes by
phosphoenolpyruvate carboxylase from C4 plants. Plant Physiol 59: 564-568
29. RIvERA ER, BN SMITH 1979 Crystal morphology and 13C/'2C composition of
solid oxalate in cacti. Plant Physiol 64: 966-970
30. SCHMIDT H-L, FJ WINKLER 1979 Einige Ursachen der Variations breite von 13CWerten bei C3- und C4-Pflanzen. Ber Deutsch Bot Ges 92: 185-191
31. SMITH BN, S EPSTEIN 1971 Two categories of I3C/2C ratios for higher plants.
Plant Physiol 47: 380-384
32. TROUGHTON JH 1979 6`3C as an indicator of carboxylation reactions, In M
Gibbs, E Latzko, eds, Encyclopedia of Plant Physiology, New Series Vol 6,
Springer-Verlag, Heidelberg, pp 140-149
33. WHELAN T, WM SACKETT, CR BENEDICT 1970 Carbon isotope discrimination in
a plant possessing the C4 dicarboxylic acid pathway. Biochem Biophys Res
Commun 41: 1205-1210
34. WHELAN T, WM SACKETT, CR BENEDICT 1973 Enzymatic fractionation ofcarbon
isotopes by phosphoenolpyruvate carboxylase from C4 plants. Plant Physiol
51: 1051-1054
35. WONG SC, IR COWAN, GD FARQUHAR 1978 Leaf conductance in relation to
assimilation in Eucalyptuspauciflora sieb ex spreng. Plant Physiol 62: 670-674
36. WONG SC, IR COWAN, GD FARQUHAR 1979 Stomatal conductance correlates
with photosynthetic capacity. Nature 282: 424-426
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1980 American Society of Plant Biologists. All rights reserved.