Determination of Nitrate Uptake and Accumulation Using ¹⁵N in Rice Seedlings

Materials and Reagents

1. Rice seedlings: Four weeks old seedlings
   
2. NH₄NO₃ (catalog number: 6484-52-2 )
   
3. KH₂PO₄ (catalog number: 7778-77-0 )
   
4. K₂SO₄ (catalog number: 7778-80-5 )
   
5. CaCl₂^.2H₂O (catalog number: 94248-52-9)
   
6. MgSO₄^.7H₂O (catalog number: 10034-99-8 )
   
7. Na₂SiO₃ (catalog number: 1344-09-8 )
   
8. NaFeEDTA (catalog number: 7720-78-7; 139-33-3)
   
9. H₃BO₃ (catalog number: 10043-35-3 )
   
10. MnCl₂^.4H₂O (catalog number: 20603-88-7)
    
11. CuSO₄^.5H₂O (catalog number: 7758-99-8 )
    
12. ZnSO₄^.7H₂O (catalog number: 7446-20-0 )
    
13. Na₂MoO₄^.2H₂O (catalog number: 7631-95-0 )
    
14. CaSO₄^.2H₂O (catalog number: 10101-41-4 )
    
15. IRRI nutrient solution (see Recipes)
    
16. 0.1 mM CaSO₄ solution (see Recipes)
    
17. K¹⁵NO₃ (catalog number: 57654-83-8 ) (see Recipes)
    

Equipment

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

Procedure

1. 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%.
   
2. 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.
   
3. 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.
   
4. 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.
   
5. Influx or accumulation of ¹⁵NO₃^- was calculated from the ¹⁵N concentrations of the roots.
   

Recipes

1. 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
   
2. 0.1 mM CaSO₄ solution (1 L)
   0.0172 g CaSO₄^.2H₂O
   Add ddH₂O to final volume
   
3. K¹⁵NO₃
   0.5 mM K¹⁵NO₃
   

Acknowledgments

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

References

1. 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. 
2. 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.