Summary

 

Authors

Margaret Barbour, Lucas Cernusak

Definition

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 N_t is the amount of N present in the sample, N_l and N_u 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:

Measurement approaches

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%
		Second flush	18%	56%
Vaccinium vitis-idaea	evergreen	First flush	47%	64%
		Second flush	10%	25%

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.

Related techniques

Natural abundance ¹⁵N

Literature references

Grelet et al. (2001) Journal of Experimental Botany, 52, 993-1002.

Millard & Neilsen (1989) Annals of Botany, 63, 301-309.