Materials and Reagents

Rice seedlings: Four weeks old seedlings
NH₄NO₃ (catalog number: 6484-52-2 )
KH₂PO₄ (catalog number: 7778-77-0 )
K₂SO₄ (catalog number: 7778-80-5 )
CaCl₂^.2H₂O (catalog number: 94248-52-9)
MgSO₄^.7H₂O (catalog number: 10034-99-8 )
Na₂SiO₃ (catalog number: 1344-09-8 )
NaFeEDTA (catalog number: 7720-78-7; 139-33-3)
H₃BO₃ (catalog number: 10043-35-3 )
MnCl₂^.4H₂O (catalog number: 20603-88-7)
CuSO₄^.5H₂O (catalog number: 7758-99-8 )
ZnSO₄^.7H₂O (catalog number: 7446-20-0 )
Na₂MoO₄^.2H₂O (catalog number: 7631-95-0 )
CaSO₄^.2H₂O (catalog number: 10101-41-4 )
IRRI nutrient solution (see Recipes)
0.1 mM CaSO₄ solution (see Recipes)
K¹⁵NO₃(catalog number: 57654-83-8 ) (see Recipes)

Equipment

Isotope Ratio Mass Spectrometer (Thermo Fisher Scientific, model: MAT253 )
Elemental Analyzer (Thermo Fisher Scientific, model: Flash EA1112 HT )

Procedure

The rice seeds were surface sterilized with 10% (v/v) hydrogen peroxide for 30 min and then rinsed thoroughly with deionized water. The sterilized seeds were germinated on plastic supporting netting (mesh of 1 mm²) mounted in plastic containers for 1 week. Uniform seedlings were selected and then transferred to a tank containing 7 L of International Rice Research Institute (IRRI) nutrient solution for 4 weeks and then deprived of N (IRRI nutrient solution without NH₄NO₃) for 1 week. All the plants were grown in a growth room with a 16-h-light (30 °C)/8-h-dark (22 °C) photoperiod, and the relative humidity was controlled at approximately 70%.
The plants were transferred first to a container with 7 L washing solution (0.1 mM CaSO₄) for 1 min, then to a new container with 7 L complete nutrient solution containing 0.5 mM K¹⁵NO₃ (atom% ¹⁵N: 80.25%) for 5 min uptake, and finally to washing solution (0.1 mM CaSO₄) again for 1 min. Make sure the whole root system were socked in the solution.
For analyzing the nitrate accumulation, the N-starved seedlings were transferred to an IRRI nutrient solution containing 0.5 mM K¹⁵NO₃ (atom% ¹⁵N: 80.25%) for 24 h before the harvest.
Harvest the roots and shoots respectively and grinding in liquid N, the powder was dried to a constant weight at 70 °C. About 10 mg of powder of each sample was analyzed using the Isotope Ratio Mass Spectrometer system.
Influx or accumulation of ¹⁵NO₃^- was calculated from the ¹⁵N concentrations of the roots.

Recipes

IRRI nutrient solution
1.25 mM NH₄NO₃
0.3 mM KH₂PO₄
0.35 mM K₂SO₄
1 mM CaCl₂^.2H₂O
1 mM MgSO₄^.7H₂O
0.5 mM Na₂SiO₃
20 μM NaFeEDTA
20 μM H₃BO₃
9 μM MnCl₂^.4H₂O
0.32 μM CuSO₄^.5H₂O
0.77 μM ZnSO₄^.7H₂O
0.39 μM Na₂MoO₄^.2H₂O
pH 5.5
0.1 mM CaSO₄ solution (1 L)
0.0172 g CaSO₄^.2H₂O
Add ddH₂O to final volume
K¹⁵NO₃
0.5 mM K¹⁵NO₃

Acknowledgments

This protocol is adapted from Delhon et al. (1995) and Tang et al. (2012).

References

Delhon, P., Gojon, A., Tillard, P. and Passama, L. (1995). Diurnal regulation of NO₃^- uptake in soybean plants I. Changes in NO₃^- influx, efflux, and N utilization in the plant during the day/night cycle. J Exp Bot 46(10): 1585-1594.
Tang, Z., Fan, X., Li, Q., Feng, H., Miller, A. J., Shen, Q. and Xu, G. (2012). Knockdown of a rice stelar nitrate transporter alters long-distance translocation but not root influx. Plant Physiol 160(4): 2052-2063.