Margaret Barbour, Lucas Cernusak
15N-enriched substrates can be used to experimentally trace nitrogen uptake and remobilization by plants. The source of plant N is carefully controlled, usually by hydroponic techniques, and a switch from natural abundance 15N to enriched 15N is imposed at a relevant developmental stage. After growth under the 15N enriched N source, the plant tissue is harvested and analysed for 15N and %N on an isotope ratio mass spectrometer.
Terminology and equations
The amount of labeled N is distinguished from unlabelled N using (Grelet et al. 2001):
where Nt is the amount of N present in the sample, Nl and Nu are the amounts of labeled N and unlabelled N, respectively, and , 0 and 1 are the 15N abundance (in atom %) of the sample, the plant tissue prior to the start of labeling, and the nutrient solution, respectively. Atom % is calculated:
Laboratories will usually provide 15N compositions in per mil (‰) relative to the standard. 15N and %N of a sample may be converted to Atom% by:
When assessing remobilization of -old’ (unlabelled) N and allocation of -new’ (labelled) N, it is often useful to divide the harvested plants into physiologically-meaningful tissues. For example, Millard and Neilsen (1989) divided their apple trees into leaves, stems, new stem growth, roots, and fine roots sieved from the growth medium. These tissues were freeze-dried and milled before being analysed separately for %N and 15N. In the case of an evergreen plant, or an experiment in which the switch from unlabelled to labeled N occurred when live leaves were present, it may be more meaningful to divide the leaves and roots into old and new portions.
The form of 15N labeling applied may need to be tailored to suit the N requirements of the plant species considered. In a number of studies 15N has been supplied as doubly-labeled 15NH415NO3.
Ranges of values
Using this approach, one may estimate the proportion of unlabeled and labeled N in each plant tissue. If the plant tissue was formed after the switch to labeled N, then the proportion of unlabeled N must be derived from N remobilized from older tissues. An example of values for the proportion of remobilized N in new leaves for a deciduous and an evergreen tree species may be found in Grelet et al. (2001). In summary:
|Species||Habit||%N from remobilization||High N||Low N|
|Vaccinium myrtillus||deciduous||First flush||72%||87%|
|Vaccinium vitis-idaea||evergreen||First flush||47%||64%|
Health, safety and hazardous waste disposal considerations
NH4NO3 is classed as a hazardous substance as it can be explosive (class 5.1 oxidising agent). However, it is stable in solid form so long as it does not come into contact with incompatible substances (chlorides and metals such as chromium, copper, cobalt and nickel), and is not exposed to high temperatures or strong shocks. A Material Safety Data Sheet (MSDS) must be obtained from the supplier, and kept in a register close to where people who may be exposed can refer to the MSDS easily.
Natural abundance <sup>15</sup>N
Grelet et al. (2001) Journal of Experimental Botany, 52, 993-1002.
Millard & Neilsen (1989) Annals of Botany, 63, 301-309.