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Nitrogen-15 is a rare stable isotope of nitrogen. This isotope is often used in agricultural research. For example, Nitrogen-15 is used to trace mineral nitrogen compounds and translocate the nitrogen molecule in plants. This protocol is used to determine nitrate uptake and accumulation in rice seedlings by using Nitrogen-15.

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

Plant Science > Plant physiology > Nutrition
Authors: Zhong Tang
Zhong TangAffiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement and Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China
Bio-protocol author page: a783
 and Guohua Xu
Guohua XuAffiliation: State Key Laboratory of Crop Genetics and Germplasm Enhancement and Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, Nanjing, China
For correspondence: ghxu@njau.edu.cn
Bio-protocol author page: a784
Vol 3, Iss 16, 8/20/2013, 2023 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.875

[Abstract] Nitrogen-15 is a rare stable isotope of nitrogen. This isotope is often used in agricultural research. For example, Nitrogen-15 is used to trace mineral nitrogen compounds and translocate the nitrogen molecule in plants. This protocol is used to determine nitrate uptake and accumulation in rice seedlings by using Nitrogen-15.

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.


How to cite: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tang, Z. and Xu, G. (2013). Determination of Nitrate Uptake and Accumulation Using 15N in Rice Seedlings. Bio-protocol 3(16): e875. DOI: 10.21769/BioProtoc.875; Full Text
  2. Tang, Z., Fan, X., Li, Q., Feng, H.,Miller, A. J., Shen, Q. and Xu, G. (2012). Knockdownof a rice stelar nitrate transporter alters long-distance translocation but notroot influx. Plant Physiol 160(4): 2052-2063.




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