Summary

Author

Lucas Cernusak

Definition

Plant ¹⁵N is a measurement of the stable nitrogen isotope ratio of plant material. The two stable isotopes of nitrogen are ¹⁵N and ¹⁴N. In nature, ¹⁵N makes up approximately 0.4% of nitrogen, whereas ¹⁴N makes up approximately 99.6%. The ¹⁵N has an extra neutron compared to ¹⁴N.

Terminology and equations

¹⁵N is defined as,

,

where R_X is the ¹⁵N/¹⁴N ratio of a sample and R_Std is the ¹⁵N/¹⁴N ratio of a standard. The international standard for ¹⁵N is N₂ in air. This standard has a ¹⁵N/¹⁴N ratio of 0.003676. The ¹⁵N thus expresses the proportional departure of the ¹⁵N/¹⁴N ratio of a sample from this reference value. Because the departures are generally very small for natural abundance measurements, the ¹⁵N is typically multiplied by 1000, so that it is given as per mil, or per 1000. The per mil symbol is ‰.

Measurement approaches

Natural abundance nitrogen isotope ratios are typically measured on an isotope ratio mass spectrometer. To measure the ¹⁵N of plant tissue, the plant tissue should be dried to a constant mass at 60 to 80^∘C in a drying oven. Alternatively, the sample can be lyophilized. It should then be ground to a fine, homogenous powder (roughly resembling the texture of flour). This will ensure that the small subset of tissue on which the measurement is made will be representative of the tissue as a whole. The analysis is then typically conducted on 1 to 3 mg of dried plant powder, although larger samples may be required for tissues such as wood that have low nitrogen concentrations. The subsample of dried plant powder is placed into a tin capsule, which is loaded into the auto-sampler of an elemental analyzer connected to an isotope ratio mass spectrometer, by way of a continuous flow interface. The plant material is combusted, and then passes through a reduction column, which converts all nitrogen to N₂. The N₂ then enters the mass spectrometer, and the ratio of ¹⁵N/¹⁴N in the N₂ is determined.

For some applications, an investigator may be interested in ascertaining the ¹⁵N of a specific compound or metabolite in the plant. This generally requires that the compound of interest be extracted from the bulk plant biomass. In this case, care must be taken that the nitrogen isotope ratio of the target compound is not altered during the extraction process. Once the compound is isolated, it can be analyzed as described above for bulk plant tissues.

Ranges of values

Plant ¹⁵N values at natural abundance can range from approximately -15‰ to 20‰. Variation in plant ¹⁵N can be caused both by variation in ¹⁵N of the plant’s nitrogen sources and by discrimination against ¹⁵N during nitrogen uptake.

Atmospheric nitrogen fixation through bacterial symbioses generally shows little discrimination with respect to atmospheric N₂. Thus, nitrogen acquired from the atmosphere, for example by nitrogen-fixing legumes, should have a ¹⁵N close to 0‰.

Discrimination against ¹⁵N during uptake of nitrate or ammonium from the soil solution has mostly been observed at high external nitrate and ammonium concentrations. Under these conditions, there can be a significant efflux of nitrate or ammonium from the root cells. This efflux represents a branch point in the flux pathway, and therefore allows the possibility for discrimination against ¹⁵N. Both nitrate reductase and glutamine synthetase discriminate against ¹⁵N, with discrimination constants of approximately 15‰ and 17‰, respectively. When nitrate or ammonium efflux rates from root cells are low, the discrimination expressed by these enzymes will also be low. When efflux rates from root cells are very high, on the other hand, discrimination by these enzymes will approach the values of the discrimination constants given above. In the case of nitrate, the potential for discrimination by nitrate reductase can be effectively cancelled if nitrate is loaded into the xylem and assimilated in leaves. Nitrate loading into the xylem is assumed not to discriminate against ¹⁵N. Additionally, nitrate is not exported from leaves in the phloem, except in trace amounts. Thus, when nitrate is loaded into the xylem in the roots, the potential branch point in the flux pathway (whereby nitrate would be lost from the plant) is effectively removed.

Mycorrhizal plants generally have lower ¹⁵N than non-mycorrhizal plants growing under similar conditions, suggesting that mycorrhizal associations decrease plant ¹⁵N. The mechanisms by which this occurs are not well understood.

Health, safety and hazardous waste disposal considerations

There are no specific health and safety risks associated with sampling for plant ¹⁵N.

Related techniques

See associated sections on:

Soils and Rhizosphere

Leaf nitrogen (N) concentration and leaf phosphorus (P) concentration

Determining Available Nitrogen

Literature references

Comstock JP (2001) Steady-state isotopic fractionation in branched pathways using plant uptake of NO₃^– as an example. Planta 214, 220-234.

Craine JM, Elmore AJ, et al. (2009) Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability. New Phytologist 183, 980-992.

Evans RD (2001) Physiological mechanisms influencing plant nitrogen isotope composition. Trends in Plant Science 6, 121-126.

Handley LL, Raven JA (1992) The use of natural abundance of nitrogen isotopes in plant physiology and ecology. Plant, Cell and Environment 15, 965-985.

Hogberg P (1997) ¹⁵N natural abundance in soil-plant systems. New Phytologist 137, 179-203.

Pate JS, Stewart GR, Unkovich M (1993) ¹⁵N natural abundance of plant and soil components of a Banksia woodland ecosystem in relation to nitrate utilization, life form, mycorrhizal status and N₂-fixing abilities of component species. Plant, Cell and Environment 16, 365-373.

Robinson D (2001) ¹⁵N as an integrator of the nitrogen cycle. Trends in Ecology & Evolution 16, 153-162.