STUDIA UNIVERSITATIS BABES-BOLYAI, PHYSICA, SPECIAL ISSUE, 2003
PHOTOSYNTHETIC FRACTIONATION OF CARBON ISOTOPES
Stela Cuna1, Gabriela Muresan1, Onuc Cozar2, Nicolae Lupsa1, Valentin
Mirel1
1. National Institute of Research and Development for
Isotopic and Molecular Technologies Cluj Napoca
2. Babes-Bolyai University Cluj Napoca
ABSTRACT. Variation in carbon isotopic composition among plant
species could be used to distinguish among photosynthetic pathway types,
to determine the water-use efficiency, and the adaptation of the plant at
the environmental stress. The method that we have proposed to establish
the photosynthetic fractionation from the plants consists of two stages. In
the first stage the organic matter is converted to CO2 by dry combustion
in an excess of oxygen. In the second stage the stable carbon isotope
ratios (13C/12C) is measured with high precision by mass spectrometry
with double collector. We have analyzed the carbon isotopic composition
of five plants (Malus domestica, Zea mays, Robinia pseudoacacia, Juglans
regia and Castanea sativa), and we have determined the linearity and
precision of the method, and the most propitious size of the sample to be
analyzed. The precision was ± 0.2 ‰ for 10 mg of dry plant used for
combustion. The photosynthetic pathway for these plants was different,
and we have joined them with C3 and C4 type of the photosynthesis.
1. Introduction
The use of carbon isotope ratios in plant ecological and physiological
research has increased significantly [1]. The two stable isotopes of carbon ( 13C and
12
C) are not equally distributed in natural compounds because of isotope
fractionation occurring during physical, chemical and biological processes
involved in the carbon cycle. Normally plants are depleted in 13C compared to
atmospheric CO2 because of carbon isotope fractionation occurring during
photosynthetic CO2 fixation. Carbon isotope discrimination () is a measure of this
process and depends on fractionation during diffusion and during enzymatic
carboxylation reactions. In plants possessing C3 photosynthetic pathways the major
components contributing to the overall fractionation are the differential diffusibility
of CO2 across the stomatal pathway and the fractionation by Rubisco carboxylase
(13C range between -35‰ and -22‰). Variation in composition among plants
with the C4 photosynthetic pathway is less than in C3 plants (13C from -20‰ to 8‰) because there is another carboxylase (PEP carboxylase) that fixed carbon
during photosynthesis [2]. Then, carbon isotope abundance becomes a classical
means to distinguish between different photosynthetic pathways and to study their
geographical, taxonomic and ecological distributions.
STELA CUNA, GABRIELA MURESAN, O. COZAR, N. LUPSA, V. MIREL
2. Materials and methods
Carbon isotope discrimination was analyzed in the leaves of five plant
species: Malus domestica, Robinia pseudoacacia, Julans regia, Castanea sativa, and
Zea mays.
Sample collection was straightforward. We have collected the samples at
the same time of the day, because diurnal changes in starch and sugar contents can
affect carbon isotope ratio. To further minimize sample variation in populationlevel studies, samples were collected from the same canopy position. There can be
13Cair gradients that could potentially confound interpretation of plant isotopic
values, especially in dense canopies. After harvesting, the five plant samples were
dried immediately at a 700C to avoid loss of organic materials. Samples were
stored at room temperature after drying for later determination of their isotopic
composition.
Because of instrumental requirements, carbon from plant must be
converted to CO2 for stable isotope ratio measurements. Conversion of organic
samples (plant’s leaves) to CO2 for isotopic analysis is accomplished by dry
combustion in an excess of oxygen. We have chosen the simplest and fastest
method that involves the combustion of individual sample in sealed quartz tubes.
Because each sample is prepared in its own container, there is no chance for
memory effects. For combustion we used 3 mg, 5 mg, 7 mg, and 10 mg of dried
leaves from each five plants. The dried tissues have grounded to pass through a 40mesh screen [3].
The materials needed for this method was: quartz tubes for combustion and
for sample boats, CuO as the oxygen source, furnace, and a vacuum system for
purification and measurement of CO2 generated by combustion
The CO2 was separated from the other combustion products by cryogenic
distillation. We used a simple vacuum line who consists of a diffusion pump to
provide a high vacuum, a tube cracker for breaking the combustion tube under
vacuum, a purification trap for cryogenic distillation of the combustion products, a
manometer for measuring the volume of CO2 produced, and a port for attaching
sample bulbs.
CO2 purified was analyzed with a mass spectrometer equipped with a
double inlet system and double collector, type Atlas 86 designed by Varian MAT.
The CO2 gas generated from the sample was admitted to the sample side of the
dual-inlet system, and a reference gas of known carbon isotope composition was
admitted to the standard side of the inlet. Then, sample and standard gases were
alternately admitted to the ion source of the mass spectrometer, and the isotopic
composition of the sample was repeatedly and rapidly compared with that of the
standard.
PHOTOSYNTHETIC FRACTIONATION OF CARBON ISOTOPES
3. Results and discussions
The results of the measurements are presented in Table 1, Table 2 and
Fig.1.
Sample
size
3mg
5mg
7mg
10mg
The variation of 13 with sample size
Malus
Castanea
Robinia
domestica
sativa
pseudoacacia
13C ‰
13C ‰
13C ‰
-27.02
-26.63
-26.20
-26.57
-27.27
-26.91
-27.52
-25.28
-26.66
-27.49
-25.30
Table 1
Juglans
regia
13C ‰
-25.54
-26.62
-26.12
Zea mays
13C ‰
-10.17
-10.03
-9.60
-9.25
We have taken 3 mg, 5 mg, 7 mg and 10 mg from each plant to see the
variation of carbon isotopic composition (13C) with sample size (Table 1). The
results show this variation is about 1‰ for any plant. This means that it is possible
that some errors can appear during sample preparation because biochemical
heterogeneity within the sample. Heterogeneity in ground leaves arises because of
incomplete grinding and mining and small differences in metabolic activity among
different leaves in a bulked sample.
The amount of sample required will depend largely on volume of the mass
spectrometer inlet system and carbon content of the sample. We have think that
sample size of 5 mg for plant tissue should be adequate. In Fig. 1 there is one
examples of variation of the 13C with sample size.
-3 0
-2 9
8
-2 8
6
-2 7
4
-2 6
2
D e lta 1 3 C ( ‰ )
S a m p le s iz e ( m g )
1 0
-2 5
0
-2 4
1
2
3
4
C a s ta n e a s a tiv a
Fig.1. The variation 13C with sample size in Castanea sativa
The variation 13C between C3 and C4 plants is shows in Table 2. The 1-4
plants with an average 13C= -26.48‰ are C3 plants. In this group of plants the
isotopic fractionation of carbon in leaves is caused by ribulose bisphosphate
carboxylase (Rubisco) [1], [2]. Zea mays belongs to C4 plants because for this plant
STELA CUNA, GABRIELA MURESAN, O. COZAR, N. LUPSA, V. MIREL
13C is -9.76‰ and this means that isotopic fractionation of carbon is caused by
PEP carboxylase.
No.
1
2
3
4
5
The variation 13Cvs. PDB of studied plants
Sample
Photosynthetic
13Cvs. PDB [‰]
group
Malus domestica
-26.86
C3
Castanea sativa
-27.06
C3
Robinia pseudoacacia
-26.01
C3
Juglans regia
-26.01
C3
Zea mays
-9.76
C4
Table 2
The carbon isotopic composition is approximately the same for the plants
C3 that means this studied plants had the same source of CO2 because
photosynthetic rate is linearly related to ambient CO2 concentration. Also, these
values reflect the fact that we have collected the samples at the same time of the
day. There is a little difference in 13C values between Juglans regia (nut tree) and
Castanea sativa (chestnut tree). The lower 13C value in Juglans regia indicates a
lower intercellular CO2 concentration because the species of nut trees occurs on
much drier site those chestnut trees.
3. Conclusions
We have analyzed the carbon isotope composition of five plant species by
isotope ratio mass spectrometry (IRMS). Carbon was extracted from leaves as CO 2
by combustion and purified by cryogenic distillation. The precision of
measurements was 0.2‰ for 10 mg of dry plant used for combustion. The
photosynthetic pathway of the studied plants was different, and we have joined
Malus domestica, Robinia pseudoacacia, Juglans regia and Castanea sativa with C3
type of photosynthesis and Zea mays with C4 type of the photosynthesis.
The results obtained are in good agreement with the model predicts in
literature.
4. References
[1] E. B r ug no li , G. D. Fa r q u ha r "Photosynthetic fractionation of carbon isotopes" in
"Photosynthesis: Physilogy and Metabolism", pp. 399-434, R. C. Leegood, T.D. Sharkey
and S. von Caemmerer 9eds.), Kluwer Academic Publishers, The netherlands, 2000
[2] G. D. F ar q u har , J . R . E h ler i n ger, , K. T . Hub ic k, "Carbon isotope
discrimination and photosynthesis, Annu. Rev. Plant physiol. Plant Mol.Biol., 40, 503-537,
1989
[3] T . W . B o utto n "Stable carbon Isotope Ratios of Natural materials: Sample
preparation and Mass Spectrometric Analysis" in Carbon Isotope Techniques, pp. 155-170,
D. C. Coleman, B. Fry (eds.), Academic Press Inc., San Diego, 1991