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Measurement of Net NO3- Flux in Rice Plants with the SIET System
水稻NO3-吸收量的测定-SITE法(scanning ion-electrode technique)   

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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(硝酸盐转运蛋白), Root(根), SIET system(SIET系统), Nitrate flux(硝酸盐通量), Rice(大米)

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.

简介

SIET(扫描离子电极技术)是一种新的技术,通过使用微电极和微传感器在活生物材料中实时研究离子和分子的流速。 该技术允许在完整植物的表面上非侵入性地同时测量特定离子的通量。 它具有高时间和空间分辨率。 该协议使用SIET系统测量水稻植物中的离子通量率。

关键字:硝酸盐转运蛋白, 根, SIET系统, 硝酸盐通量, 大米

材料和试剂

  1. 水稻幼苗:三周龄幼苗
  2. 塑料支撑网
  3. 三丁基氯硅烷(Fluka,目录号:90796)
  4. NH 4 3
  5. KH 2 PO 4
  6. K 2 SO 4
  7. CaCl 2 2H O
  8. MgSO 4。 。 O
  9. Na SiO 3
  10. NaFeEDTA
  11. H 3 BO 3
  12. MnCl 2 4H O
  13. CuSO 4 5H sub 2 O
  14. ZnSO 4 。 7H O
  15. Na <2> MoO <4> 2H O
  16. 国际水稻研究所(IRRI)营养液(见配方)
  17. 校准溶液(参见配方)
  18. 测量溶液(参见配方)

设备

  1. 小塑料盘(直径6厘米)
  2. 测量室
  3. 离子选择性电极(Clark Electromedical,型号:GC150-10)
  4. 具有ASET 2.0的BIO-IM(NMT-YG-100,Younger USA LLC,Amherst,型号:MA01002)

软件

  1. ASET 2.0(Sciencewares,Falmouth,目录号:MA 02540)
  2. iFluxes 1.0(YoungerUSA,LLC,Amherst,目录号:MA 01002)软件

程序

  1. 将水稻种子用10%(v/v)过氧化氢表面灭菌30分钟,然后用去离子水彻底冲洗。
  2. 将灭菌的种子在安装在塑料容器中的塑料支撑网(1mm筛目)上在具有16小时光照(30℃)/8小时的生长室中发芽1周(22℃)光周期,相对湿度控制在约70%。选择均一的幼苗,然后转移到IRRI营养液中。
  3. 水稻幼苗在IRRI营养液中生长2周,然后剥夺N(没有NH 4 NO 3的IRRI营养液)3天。所有植物在具有16小时光照(30℃)/8小时暗(22℃)光周期的生长室中生长,并且相对湿度控制在约70%。
  4. 在室温(24℃-26℃)下测量之前,将幼苗的根在测量溶液1(无NO 3+缓冲液)中平衡20至30分钟。
  5. 然后将平衡的幼苗转移到测量室中,并用10ml新鲜测量溶液2填充小塑料皿(直径6cm),所述新鲜测量溶液2含有0.25mM NO 3 - >。
  6. 离子选择性电极由1.5mm(外径)硼硅酸盐空白制成。将坯料拉1μm直径的尖端,然后用三丁基氯硅烷硅烷化。将电极坯料的尖端破碎成2-3μm的直径,然后用适当的溶液回填。 NO 3+的反填充溶液是0.5M KNO 3和0.1M KCl。用校准溶液1(0.05mM NO 3+)校准电极并校准溶液2(0.5mM NO 3 -/sup>),然后进行通量测量
  7. 当根部固定在培养皿的底部时,微电极在测量溶液中沿着垂直于根分生组织区的轴在距离主根表面5和35μm之间的两个位置之间振动。通过在不含根的测量溶液中振动电极记录背景。测量持续15分钟。
  8. 分析所获得的数据,并将其转换为NO 3 - 流出(阴性)(pmol cm -2 --s sup -1 - 1/sup >)使用MageFlux程序(http://www.xuyue.net/mageflux)。每种类型的转化细胞周围的离子流测定独立地重复五次

食谱

  1. IRRI营养液
    1.25mM NH 4 NO 3 sub/
    0.3mM KH 2 PO 4 4/v/v 0.35mM K 2 SO 4 4/v/v 1mM CaCl 2 2H O
    1mM MgSO 4。 7H 2 0.5mM Na 2 SiO 3子等 20μMNaFeEDTA
    20μMH sub 3 BO 3
    9μMMnCl 2 。 4H 2 O
    0.32μMCuSO 4 5H 2 0.77μMZnSO 4 sub 7H 2 O 0.39μMNa 2 MoO 4+ 2H 2 O O pH 5.5
  2. 校准溶液
    1. 校准溶液1
      (pH6.5)
      0.05mM NO 3 -
      0.025mM Ca(NO 3)2 sub。 0.1mM CaCl 2/v/v 0.1mM NaCl 0.1mM MgSO 4 0.3 mM MES
      pH 6.5
    2. 校准溶液2
      h (pH 5.5) 0.5mM NO 3 -
      0.25mM Ca(NO 3)2 sub。 0.1mM CaCl 2/v/v 0.1mM NaCl 0.1mM MgSO 4 0.3 mM MES
      pH 5.5
  3. 测量溶液
    1. 测量溶液1
      0.2mM CaCl 2·h/v 0.1mM NaCl 0.1mM MgSO 4 0.3 mM MES
      pH 6.0
    2. 测量溶液2
      0.125mM Ca(NO 3)2 sub。 0.1mM CaCl 2/v/v 0.1mM NaCl 0.1mM MgSO 4 0.3 mM MES
      pH 6.0

致谢

该协议改编自Xu et al。(2006); Sun (2009); 和Tang等人(2012)。

参考文献

  1. Sun,J.,Chen,S.,Dai,S.,Wang,R.,Li,N.,Shen,X.,Zhou,X.,Lu,C.,Zheng,X.,Hu, Zhang,Z.,Song,J.and Xu,Y。(2009)。 NaCl诱导的耐盐和耐盐的根中细胞和组织离子通道的交替, 敏感的杨树种类。 植物生理 149(2):1141-1153。
  2. Tang,Z.,Fan,X.,Li,Q.,Feng,H.,Miller,A.J.,Shen,Q.and Xu,G。(2012)。 敲除大米硝酸盐转运蛋白可改变长距离易位,但不会改变根系流入。 植物生理学 160(4):2052-2063。
  3. Xu,Y.,Sun,T.and Yin,L.P。(2006)。 应用非侵入性微型传感系统同时测量 H + 和O 2 通量在花粉管周围。植物生物学 48(7):823-831。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: 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.
  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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