Photosynthetic Fractionation of Carbon Isotopes

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 12C) 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 13 the C4 photosynthetic pathway is less than in C3 plants ( C 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 population- level studies, samples were collected from the same canopy position. There can be 13  Cair 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 40- mesh 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. Table 1 The variation of 13 with sample size Sample Malus Castanea Robinia Juglans Zea mays size domestica sativa pseudoacacia regia 13C ‰ 13C ‰ 13C ‰ 13C ‰ 13C ‰ 3mg -27.02 -26.63 -26.20 -25.54 -10.17 5mg - -26.57 -27.27 -26.62 -10.03 7mg -26.91 -27.52 -25.28 - -9.60 10mg -26.66 -27.49 -25.30 -26.12 -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.

1 0 - 3 0 ) g - 2 9 m 8 ) (

- 2 8 ( z

i 6 s C

e - 2 7

p 4 a t m - 2 6 l a e S 2 D - 2 5

0 - 2 4 1 2 3 4 C a s t a n e a s a t i v a

Fig.1. The variation 13C with sample size in Castanea sativa 13 The variation  C between C3 and C4 plants is shows in Table 2. The 1-4 13 plants with an average  C= -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. Table 2 13 The variation  Cvs. PDB of studied plants 13 No. Sample  Cvs. PDB [‰] Photosynthetic group

1 Malus domestica -26.86 C3 2 Castanea sativa -27.06 C3 3 Robinia pseudoacacia -26.01 C3 4 Juglans regia -26.01 C3 5 Zea mays -9.76 C4

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. Brugnoli, G. D. Farquhar "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. Farquhar, J. R. Ehleringer, , K. T. Hubick, "Carbon isotope discrimination and photosynthesis, Annu. Rev. Plant physiol. Plant Mol.Biol., 40, 503-537, 1989

[3] T. W. Boutton "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