Measurement of Net NO₃^- Flux in Rice Plants with the SIET System

Materials and Reagents

1. Rice seedlings: Three weeks old seedlings
   
2. Plastic supporting netting
   
3. Tributylchlorosilane (Fluka, catalog number: 90796 )
4. NH₄NO₃
   
5. KH₂PO₄
   
6. K₂SO₄
   
7. CaCl₂^.2H₂O
   
8. MgSO₄^.7H₂O
   
9. Na₂SiO₃
   
10. NaFeEDTA
    
11. H₃BO₃
    
12. MnCl₂^.4H₂O
    
13. CuSO₄^.5H₂O
    
14. ZnSO₄^.7H₂O
    
15. Na₂MoO_4^.2H₂O
    
16. International Rice Research Institute (IRRI) nutrient solution (see Recipes)
    
17. Calibrate solution (see Recipes)
    
18. Measuring solution (see Recipes)
    

Equipment

1. Small plastic dish (6 cm diameter)
   
2. Measuring chamber
   
3. Ion-selective electrodes (Clark Electromedical, model: GC150-10 )
   
4. BIO-IM (NMT-YG-100, Younger USA LLC, Amherst, model: MA01002 ) with ASET 2.0
   

Software

1. ASET 2.0 (Sciencewares, Falmouth, catalog number: MA 02540)
   
2. iFluxes 1.0 (YoungerUSA, LLC, Amherst, catalog number: MA 01002) software
   

Procedure

1. Rice seeds were surface sterilized with 10% (v/v) hydrogen peroxide for 30 min and then rinsed thoroughly with deionized water.
   
2. The sterilized seeds were germinated on plastic supporting netting (mesh of 1 mm²) mounted in plastic containers for 1 week 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%. Uniform seedlings were selected and then transferred to IRRI nutrient solution.
   
3. Rice seedlings were grown in IRRI nutrient solution for 2 weeks and then deprived of N (IRRI nutrient solution without NH₄NO₃) for 3 d. 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%.
   
4. The roots of seedlings were equilibrated in measuring solution 1 (without NO₃^-) for 20 to 30 min before measuring at room temperature (24 °C–26 °C).
   
5. The equilibrated seedlings were then transferred to the measuring chamber, and a small plastic dish (6 cm diameter) was filled with 10 ml of fresh measuring solution 2 containing 0.25 mM NO₃^-.
   
6. Ion-selective Electrodes were made from 1.5 mm (external diameter) borosilicate blanks. The blanks were pulled to < 1 μm diameter tips using a vertical pipette puller and then silanized with tributylchlorosilane. The tips of electrode blanks were broken to a diameter of 2–3 μm, and then back-filled with appropriate solutions. The back-filling solutions for NO₃^- were 0.5 M KNO₃ and 0.1 M KCl. The electrodes were calibrated with calibrate solution 1 (0.05 mM NO₃^-) and calibrate solution 2 (0.5 mM NO₃^-) prior to flux measurements.
   
7. When the root became immobilized at the bottom of the dish, the microelectrode was vibrated in the measuring solution between two positions, 5 and 35 μm from the primary root surface, along an axis perpendicular to the root meristem zone. The background was recorded by vibrating the electrode in measuring solution not containing roots. The measuring lasted for 15 min.
   
8. The data obtained were analyzed and converted into NO₃^- influx (negative) (pmol cm^-2s^-1) using the MageFlux program (http://www.xuyue.net/mageflux). The ion flux assay around each type of transformed cells was replicated independently five times.
   

Recipes

1. IRRI nutrient solution
   1.25 mM NH₄NO₃
   0.3 mM KH₂PO₄
   0.35 mM K₂SO₄
   1 mM CaCl₂^.2H₂O
   1 mM MgSO_4^.7H₂O
   0.5 mM Na₂SiO₃
   20 μM NaFeEDTA
   20 μM H₃BO₃
   9 μM MnCl₂^.4H₂O
   0.32 μM CuSO_4^.5H₂O
   0.77 μM ZnSO₄^.7H₂O
   0.39 μM Na₂MoO₄^.2H₂O
   pH 5.5
   
2. Calibrate solution
   
   1. Calibrate solution 1
      H⁺ (pH 6.5)
      0.05 mM NO₃^-
      0.025 mM Ca(NO₃)₂
      0.1 mM CaCl₂
      0.1 mM NaCl
      0.1 mM MgSO₄
      0.3 mM MES
      pH 6.5
      
   2. Calibrate solution 2
      H⁺ (pH 5.5)
      0.5 mM NO₃^-
      0.25 mM Ca(NO₃)₂
      0.1 mM CaCl₂
      0.1 mM NaCl
      0.1 mM MgSO₄
      0.3 mM MES
      pH 5.5
      
3. Measuring solution
   1. Measuring solution 1
      0.2 mM CaCl₂
      0.1 mM NaCl
      0.1 mM MgSO₄
      0.3 mM MES
      pH 6.0
      
   2. Measuring solution 2
      0.125 mM Ca(NO₃)₂
      0.1 mM CaCl₂
      0.1 mM NaCl
      0.1 mM MgSO₄
      0.3 mM MES
      pH 6.0
      

Acknowledgments

This protocol is adapted from Xu et al. (2006); Sun et al. (2009); and Tang et al. (2012).

References

1. Sun, J., Chen, S., Dai, S., Wang, R., Li, N., Shen, X., Zhou, X., Lu, C., Zheng, X., Hu, Z., Zhang, Z., Song, J. and Xu, Y. (2009). NaCl-induced alternations of cellular and tissue ion flues in roots of salt-resistant and salt-sensitive poplar species. Plant Physiol 149(2): 1141-1153.
   
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. 
3. Xu, Y., Sun, T. and Yin, L. P. (2006). Application of non-invasive microsensing system to simultaneously measure both H⁺ and O₂ fluxes around the pollen tube. J Plant Biol 48(7): 823-831.