¹⁵N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice

Materials and Reagents

1. 96-well plate
   
2. Rice seeds (Zhonghua11, ZH11)
   
3. NaClO [Sinopharm Chemical Reagent Co.,Ltd (SCRC), catalog number: 7681-52-9 ]
   
4. KNO₃ (SCRC, catalog number: 7757-79-1 )
   
5. K¹⁵NO₃ (Sigma-Aldrich, catalog number: 57654-83-8 )
   
6. CaCl₂ (SCRC, catalog number: 10043-52-4 )
   
7. MgSO₄·7H₂O (SCRC, catalog number: 10034-99-8 )
   
8. KH₂PO₄ (SCRC, catalog number: 7778-77-0 )
9. FeSO₄·7H₂O (SCRC, catalog number: 7782-63-0 )
10. EDTA-Na₂ (SCRC, catalog number: 6381-92-6 )
11. NaSiO₃·9H₂O (SCRC, catalog number: 13517-24-3 )
    
12. H₃BO₃ (SCRC, catalog number: 10043-35-3 )
    
13. CuSO₄·5H₂O (SCRC, catalog number: 7758-99-8 )
    
14. ZnSO₄·7H₂O (SCRC, catalog number: 7446-20-0 )
    
15. MnCl₂·4H₂O (SCRC, catalog number: 13446-34-9 )
    
16. Na₂MoO₄·2H₂O (SCRC, catalog number: 10102-40-6 )
    
17. CaSO₄·2H₂O (SCRC, catalog number: 10101-41-4 )
    
18. Modified Kimura B solution (see Recipes)
    
19. 5 mM ¹⁵N-KNO₃ (see Recipes)
    
20. 0.1 mM CaSO₄ solution (see Recipes)

Equipment

1. Growth chamber (SANYO, model: MLR-351H )
   
2. Isotope ratio mass spectrometer(Thermo Fisher Scientific, model: Finnigan Delta Plus XP) with elemental analyzer (Thermo Fisher Scientific, model: Flash EA 1112 )
   

Procedure

1. Seed germination
   Rice seeds are surface-sterilized with 2.5% sodium hypochlorite (NaClO) for 30 min and then soaked in tap water, put in an incubator chamber at 37 °C for 2 days (d), change water every 12 h till the seeds germinate.
   
2. Seedling growth
   Uniformly germinated seeds are selected and put into 96-well plates, then transferred to clear water until roots length reach 3 cm, after which transplant the seeds to modified Kimura B solution. Rice seedlings are grown in a growth chamber with a 12-h light (30 °C)/12-h dark (28 °C) photoperiod and 70% humidity for about 2 weeks. The solution is changed every day.
3. ¹⁵N-nitrate uptake assay
   After 2-week cultivation, rice seedlings are pretreated with modified Kimura B solution for 2 h, after which the rice roots are washed by tap water twice and rice seedlings are transferred to modified Kimura B solution containing 5 mM ¹⁵N-KNO₃ for 3 h.
   
4. Rice seedlings harvest
   After 3 h absorption, rice roots are rinsed with 0.1 mM CaSO₄ for 2 min to remove the ¹⁵N-NO₃^- on the root surface, then roots and shoots are harvested separately and dried at 70 °C to constant weight in paper bags. Dried samples are ground to fine powder in mortars for subsequent assay.
   
5. Calculation of nitrogen uptake
   About 0.5 mg dried powder is analyzed by isotope ratio mass spectrometer and the data of ¹⁵N content are obtained (Brand, 1996). While detecting the ¹⁵N-nitrate uptake activity, a formula [total ¹⁵N amount of whole plant (TN)/dry weight (DW) of root (DWR)/3 h] is applied to the calculation, i.e., the amount of ¹⁵N take up per unit weight of roots per unit time, total ¹⁵N amount of whole plant is derived from the sum of N amount of shoots and roots. The ratio of shoot ¹⁵N content (SN) to root ¹⁵N content (RN) is used to represent the root-to-shoot transport activity (the higher the value, the higher root-to-shoot transport activity).
   
   

Representative data

Figure 1. A 96-well plate that was cut off the bottom well-suited for the growth of rice seedlings. After being put into the 96-well plate, rice roots could grow downward into solution underneath while rice shoots could grow upward tidily along each well.


Figure 2. 10-day-old rice seedlings grown on a 96-well plate. The container under the 96-well plate is full of modified Kimura B solution, usually the container is wrapped with light-tight material (e.g., tinfoil) to protect rice roots from light.

Table1. ¹⁵N content of shoots and roots


Table2. Calculation of ¹⁵N-nitrate uptake activity and root-to-shoot transport activity

Notes

1. Uniformly germinated seeds were selected to make sure the same growing status of different lines.
   
2. The germinated seeds could be transferred to modified Kimura B solution until seminal root length was about 3 to 5 cm.
   
3. The aim of pretreating rice seedlings with modified Kimura B solution for 2 h is to make sure the rice seedlings could get back to a relatively normal physiological state before the ¹⁵N-nitrate uptake assay.
   

Recipes

1. Modified Kimura B solution
   5 mM KNO₃
   0.36 mM CaCl₂
   0.54 mM MgSO₄
   0.18 mM KH₂PO₄
   40 µM FeSO₄-EDTA
   18.8 µM H₃BO₃
   13.4 µM MnCl₂
   0.32 µM CuSO₄
   0.3 µM ZnSO₄
   0.03 µM Na₂MoO₄
   1.6 mM Na₂SiO₃
   pH 6.0
   
2. 5 mM ¹⁵N-KNO₃
   As mentioned in Procedure 3, 5 mM ¹⁵N-KNO₃ was used to replace 5 mM KNO₃ in modified Kimura B solution while other ingredients remain unchanged.
   
3. 0.1 mM CaSO₄ solution
   Dissolve 0.0172 g CaSO₄·2H₂O in 1 L deionized water
   

Acknowledgments

The hydroponic culture has been illuminated in Lin et al. (2008) and Hu et al. (2015), the ¹⁵N-nitrate uptake activity and root-to-shoot transport activity assay was cited from Hu et al. (2015). This work was supported by grants from the Ministry of Science and Technology of China (2014AA10A602-5, 2015CB755702), and the Chinese Academy of Sciences (XDA08010400).

References

1. Brand, W. A. (1996). High precision isotope ratio monitoring techniques in mass spectrometry. Journal of Mass Spectrometry 31:225-235.
   
2. 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. Journal of Experimental Botany 46(291): 1585-1594.
   
3. Hu, B., Wang, W., Ou, S. J., Tang, J. Y., Li, H., Che, R. H., Zhang, Z. H., Chai, X. Y., Wang, H. R., Wang, Y. Q., Liang, C. Z., Liu, L. C., Piao, Z. Z., Deng, Q. Y., Deng, K., Xu, C., Liang, Y., Zhang, L. H., Li, L. G. and Chu, C. C. (2015). Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nature Genetics 47(7): 834-838.
   
4. Lin, S. H., Kuo, H. F., Canivenc, G., Lin, C. S., Lepetit, M., Hsu, P. K., Tillard, P., Lin, H. L., Wang, Y. Y., Tsai, C. B., Gojon, A. and Tsay, Y. F. (2008). Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. Plant Cell 20(9): 2514-2528.