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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
15N 标记法测定水稻幼苗中硝酸盐的吸收和积累

植物科学 > 植物生理学 > 营养
作者: 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, 2492 views, 0 Q&A
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.

[Abstract]

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.

材料和试剂

  1. 水稻幼苗:四周龄幼苗
  2. NH 4(NO)3(目录号:6484-52-2)
  3. KH 2 PO 4 (目录号:7778-77-0)
  4. K 2 SO (目录号:7778-80-5)
  5. (目录号:94248-52-9)
  6. MgSO 4 .7H 2 O(目录号:10034-99-8)
  7. Na 2 SiO 3(目录号:1344-09-8)
  8. NaFeEDTA(目录号:7720-78-7; 139-33-3)
  9. H 3 BO 3 (目录号:10043-35-3)
  10. (目录号:20603-88-7)。
  11. (目录号:7758-99-8)
  12. (目录号:7446-20-0)
  13. 2H 2(目录号:7631-95-0)
  14. 2 O(目录号:10101-41-4)
  15. IRRI营养液(见配方)
  16. 0.1mM CaSO 4溶液(参见配方)
  17. K 15 NO 3 (目录号:57654-83-8)(参阅配方)

设备

  1. 同位素比质谱仪(Thermo Fisher Scientific,型号:MAT253)
  2. 元素分析仪(Thermo Fisher Scientific,型号:Flash EA1112 HT)

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。 灭菌的种子在安装在塑料容器中的塑料支撑网(1mm筛网)上发芽1周。 选择均一的幼苗,然后转移到含有7L国际水稻研究所(IRRI)营养液的槽中4周,然后剥夺N(没有NH 4 NO 3的IRRI营养液 )1周。 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%。
  2. 将植物首先转移到具有7L洗涤溶液(0.1mM CaSO 4)的容器中1分钟,然后转移到具有7L完全营养液的新容器中,所述完全营养液含有0.5mM K + 5分钟摄取,最后到洗涤溶液(0.1mM CaSO 4·7H 2 O)中的溶液(5×10 6 /孔) )再次1分钟。确保整个根系统都插在解决方案中。
  3. 为了分析硝酸盐积累,将N饥饿的幼苗转移到含有0.5mM KNO 3 NO 3(原子%<15>)的IRRI营养液中, N:80.25%)24小时。
  4. 分别收获根和芽并在液体N中研磨,将粉末在70℃下干燥至恒重。使用同位素比质谱仪系统分析约10mg的每个样品的粉末。
  5. 从根的 N浓度计算 15 - 的流入或累积。 >

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 1 mM CaCl 2 2H O 1mM MgSO 4 4次。 7H 2 O 0.5mM Na 2 SiO 3子等 20μMNaFeEDTA 20μMH sub 3 BO 3 9μMMnCl 2 。 4H 2 O 0.32μMCuSO 4 sub 5H O 0.77μMZnSO 4 sub 7H 2 O 0.39μMNa 2 MoO 4+ 2H 2 O O pH 5.5
  2. 0.1mM CaSO 4溶液(1L) 0.0172g CaSO 4 sub 。 2H O 将ddH 2 O添加到最终量
  3. K 15 NO 3 0.5mM K lt 15+/NO 3/

致谢

该协议改编自Delhon等人(1995)和Tang等人(2012)。

参考文献

  1. Delhon,P.,Gojon,A.,Tillard,P。和Passama,L。(1995)。 NO 3的日间调节 - < 在大豆植物中的摄取I.在白天/夜晚循环期间植物中NO 3+上清液的流入,流出和N利用的变化。 em> 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)。 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
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How to cite this protocol: 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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