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 RX is the 15N/14N ratio of a sample and RStd 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 ‰.
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.
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