How are isotopes affected by plant processes?

Stable isotopes are a useful tool in plant physiology and ecology because isotopic fractionation occurs during both photosynthesis and transpiration - the basic physiological processes responsible for plant growth.


Photosynthesis converts CO2 in the atmosphere to carbohydrates, the building blocks of plant material.  We can consider photosynthesis in two steps: 1) CO2 enters the leaf from the atmosphere and 2) it is fixed into carbohydrates in a process known as carboxylation.  Isotopic discrimination occurs during both steps:

In step 1, CO2 diffuses into leaves through adjustable pores on the leaf surface, or stomates.  During diffusion, fractionation occurs as the heavier 13CO2 molecules diffuse more slowly. Thus, the air outside the leaf is slightly enriched in 13CO2, and the air inside the leaf pore space is depleted in 13CO2.  The discrimination value for diffusion of CO2 is 4.4‰.

In step 2 carboxylation occurs, but the details of carboxylation are different for two major pathways of photosynthesis that are found in plants.  The most common pathway is called C3 photosynthesis, because the intermediate molecule in the process has three carbon atoms. In C3 plants, CO2 binds to the enzyme Rubisco (RuBP carboxylase,).  This enzyme preferentially binds to 12CO2 if the concentration of CO2 is high and many molecules are available.  The concentration of CO2 inside the leaf (noted as ci) depends on the rate of photosynthesis and the opening of the stomatal pores, which in turn influences isotopic discrimination.  To understand this, consider the extreme case of complete closure of the stomates, so that no additional CO2 can diffuse into the leaf.  In this case all of the CO2 present inside the pore space must be used in photosynthesis, and Rubisco has no "choice" about whether to bind to 12CO2 or 13CO2.  Therefore, discrimination by carboxylation is zero.  However, the diffusional discrimination value of 4.4‰ still applies - the carbohydrates produced inside this closed leaf will carry this signature.  Because d13C of the atmosphere is equal to about -8‰, the plant material in this hypothetical case will equal -8 - 4.4 = -12.4‰.

Conversely, consider that the stomates are fully open.  In this case ci approaches the concentration of CO2 in the atmosphere, noted as ca (ci is always lower than ca).  The discrimination by diffusion becomes insignificant, but carboxylation becomes very important.  Rubisco can now "choose" from many CO2 molecules, and will preferentially bind to 12CO2.  The carbohydrates produced by this process will carry the signature of the maximum discrimination of Rubisco, about 30‰.  Adding in atmospheric d, the biomass be -38‰.

In reality, plants fall between these extreme cases of fully closed and fully open stomates.  Typical d values for C3 plants are -21 to -35‰.

We can predict the photosynthetic discrimination of plants because the relationship between ci, ca, and discrimination has been expressed mathematically:

 13D = a + (b - a)*ci/ca

 where D is discrimination, a is the diffusional discrimination (4.4) and b is the discrimination by carboxylation (30).  Thus, the isotopic composition of the plant material contains information about ci/ca, which is controlled by the rate of photosynthesis anddegree of stomatal opening, or stomatal conductance.

 There is a less common, but still important type of photosynthesis called C4 photosynthesis, after the four carbon intermediate molecule.  This pathway arose because Rubisco will bind to oxygen as well CO2, especially under high temperatures.  This process is called photorespiration, and it is undesirable because plants cannot use oxygen for photosynthesis, and actually must release CO2 to remove oxygen from the pathway.  High CO2 concentrations inhibit photorespiration, and it is likely that C3 photosynthesis evolved when the CO2 concentration in the atmosphere was much higher than it is today. As the concentration in the atmosphere dropped and photorespiration became more problematic, a new type of photosynthesis arose.

C4 photosynthesis limits photorespiration by separating the site of CO2 fixation from the leaf pore space, where the oxygen concentration is very high. In the cells surrounding the pore space (mesophyll cells), CO2 is accepted not by Rubisco, but by a different enzyme called PEP carboxylase that does not bind to oxygen.  It is converted to the 4 carbon intermediate malate and transported to special cells that surround the plant's vascular (circulatory) system.  These cells are called bundle sheath cells.  In these cells, malate is converted back to CO2 and is fixed by Rubisco in the C3 photosynthetic pathway.  Because the concentration of CO2 in bundle sheath cells is so high, photorespiration is minimized.

So why don't all plants use C4 photosynthesis?  The extra steps of fixing, transporting, and re-fixing CO2 cost energy.  C4 plants are restricted to environments and ecosystems where they have some competitive advantage over their neighbors by expending extra energy on an alternative method of photosynthesis.  Because photorespiration is dependent on temperature, C4 plants often (but not always) occur in warm climates, where up to 50% of the carbon fixed by C3 plants can be wasted as photorespiration. Some ecosystems, such as certain grasslands, are dominated by C4 plants, while others contain only a small component of plants using the C4 pathway.

Whether a plant is a C3 or C4 type is extremely important isotopically.  Bundle sheath cells are almost impermeable to diffusion, similar to the example of closed stomates discussed above.  Recall that if all of the available CO2 is fixed by Rubisco there cannot be discrimination.  Plants vary in their capacity to seal CO2 inside the bundle sheath without leakage, which can affect discrimination.  This effect as also been described mathematically:

 13D = a + (b4 - b3f - a)*ci/ca  

 This equation is similar to the equation given for C3 plants but has some extra terms.  b4 is the discrimination of PEP carboxylation of 5.7‰, far lower than the value for Rubisco, or b3, of 30‰.  f is the fraction of CO2 that leaks out of the bundle sheath cells; this value is typically near 0.2.  Thus, we can also use isotopic discrimination to gain information on ci/ca and the factors that control it in C4 plants, which are more enriched in 13C than C3 plants. Typical d values for C4 plants are -12 to -15‰.

Oxygen isotopes in CO2 are also subject to diffusional fractionation, but once inside the leaf, oxygen in water can exchange readily with oxygen in CO2 due to the presence of an enzyme called carbonic anhydrase.  Only about 1/3 of CO2 that diffuses into a C3 leaf is actually fixed in photosynthesis - the remainder diffuses back out and influences the oxygen isotope composition of atmospheric CO2. We can calculate an "apparent" discrimination against 18O in photosynthesis of C3 plants, which is not entirely based on an actual discrimination because of the exchange with leaf water:

 18D = Ra/RA - 1 = â + cc(dc - da)/(ca-cc)  

 where Ra is the ratio of 18O/16O in atmospheric CO2, RA is the ratio of 18O/16O in the net flux of CO2 into the leaf,  and cc is the CO2 concentration in the chloroplast (which is generally lower than ci). â is average fractionation during diffusion from the air into the leaf chloroplast, the site of photosynthesis. This is dominated by the diffusion from the atmosphere into leaf pore space, which is theoretically 8.8‰ based on the differences in diffusivity between C16O2 and C18O2. However, there is some influence by other fractionation factors such CO2 entering solution, so â is more accurately about 7.4‰.

 18D is highly variable across a range of ecosystems - it can vary from -20 to +32‰. This is because da and dc, the d values for 18O/16O in the atmosphere and chloroplast, respectively, are highly variable. We will discuss the factors that control them in the next sections.


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Farquhar, G. D. 1983. On the nature of carbon isotope discrimination in C4 species. Aust J Plant Physiol 10: 205-226.

Farquhar, G. D., J. R. Ehleringer, and K. T. Hubick. 1989. Carbon isotope discrimination and photosynthesis. Annual Review of Plant Physiology and Plant Molecular Biology 40: 503-537.

Farquhar, G. D., and J. Lloyd. 1993. Carbon and oxygen isotope effects in the exchange of carbon dioxide between plants and the atmosphere. Pages 47-70 in J. R. Ehleringer, A. E. Hall, and G. D. Farquhar, eds. Stable isotopes and plant carbon-water relations. Academic Press, New York.

Farquhar, G. D., J. Lloyd, J. A. Taylor, L. B. Flanagan, J. P. Syvertsen, K. T. Hubick, S. C. Wong, and J. R. Ehleringer. 1993. Vegetation effects on the isotope composition of oxygen in atmospheric CO2. Nature 363: 439-443.

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