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SIET (scanning ion-electrode technique) is a new technique to study the flow rate of the ions and molecules in real time in living biomaterials by using microelectrodes and microsensor. This technique allows non-invasive, simultaneous measurement of fluxes of specific ions at the surface of an intact plant. It has high temporal and spatial resolutions. This protocol uses the SIET system for the measurement of ions flux rate in rice plants.

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

Plant Science > Plant physiology > Ion analysis
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, 2120 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.876

[Abstract] SIET (scanning ion-electrode technique) is a new technique to study the flow rate of the ions and molecules in real time in living biomaterials by using microelectrodes and microsensor. This technique allows non-invasive, simultaneous measurement of fluxes of specific ions at the surface of an intact plant. It has high temporal and spatial resolutions. This protocol uses the SIET system for the measurement of ions flux rate in rice plants.

Keywords: Nitrate transporter, Root, SIET system, Nitrate flux, Rice

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. Na2MoO4.2H2O
  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 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. 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.


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). Measurement of Net NO3- Flux in Rice Plants with the SIET System. Bio-protocol 3(16): e876. DOI: 10.21769/BioProtoc.876; Full Text
  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.




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