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15N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice
水稻吸收和转运N15-硝酸盐的活力实验   

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Abstract

15N is a nonradioactive heavy isotope of nitrogen, widely used for biochemical and physiological research in plants. For instance, 15N-KNO3 was used as the nitrogen source in plants in order to investigate nitrate uptake activity and transport from roots to shoots (Lin et al., 2008). Here, we describe a detailed pipeline used for labeling living rice (Oryza sativa) plants with 15N-KNO3 and determination of net nitrate uptake and transport activity, and this protocol was proved to be valid in Arabidopsis and rice (Lin et al., 2008; Hu et al., 2015).

Keywords: Nitrate uptake(硝酸盐的吸收), Nitrate transport(硝酸盐转运), Rice(水稻), Isotope(同位素)

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.

简介

N是氮的非放射性重同位素,广泛用于植物的生化和生理研究。 例如,使用N-KNO 3作为植物中的氮源,以研究硝酸盐吸收活性和从根到芽的转运(Lin等人 。, 2008)。 在这里,我们描述了用于标记具有 N-KNO 3的活水稻(>稻)植物和测定硝酸盐硝酸盐吸收的详细管道 和转运活性,并且该方案在拟南芥(Arabidopsis)和水稻中被证明是有效的(Lin等人,2008; Hu等人, 2015)。

关键字:硝酸盐的吸收, 硝酸盐转运, 水稻, 同位素

材料和试剂

  1. 96孔板
  2. 水稻种子(Zhonghua11,ZH11)
  3. NaClO [国药化学试剂有限公司(SCRC),目录号:7681-52-9]
  4. KNO 3 (SCRC,目录号:7757-79-1)

  5. (Sigma-Aldrich,目录号:57654-83-8)。
  6. CaCl 2 (SCRC,目录号:10043-52-4)
  7. MgSO 4·7H 2 O(SCRC,目录号:10034-99-8)
  8. KH sub 2 PO 4(SCRC,目录号:7778-77-0)
  9. FeSO 4 7HH 2 O(SCRC,目录号:7782-63-0)
  10. EDTA-Na 2(SCRC,目录号:6381-92-6)
  11. NaSiO 3·9H 2 O(SCRC,目录号:13517-24-3)

  12. (SCRC,目录号:10043-35-3)
  13. CuSO 4·5H 2 O(SCRC,目录号:7758-99-8)
  14. ZnSO 4·7H 2 O(SCRC,目录号:7446-20-0)
  15. MnCl 2 2·4H 2 O(SCRC,目录号:13446-34-9)
  16. Na 2 MoO 4·2H 2 O(SCRC,目录号:10102-40-6)< br />
  17. (SCRC,目录号:10101-41-4)
  18. 修改的木村B解决方案(参见配方)
  19. 5 mM 15 N-KNO <3> (见配方)
  20. 0.1mM CaSO 4溶液(参见配方)

设备

  1. 生长室(SANYO,型号:MLR-351H)
  2. 同位素比质谱仪(Thermo Fisher Scientific,型号:Finnigan Delta Plus XP)用元素分析仪(Thermo Fisher Scientific,型号:Flash EA 1112)

程序

  1. 种子发芽
    水稻种子用2.5%次氯酸钠(NaClO)表面消毒30分钟,然后浸泡在自来水中,置于37℃的培养箱中2天(d),每12小时更换一次水直到种子发芽。
  2. 幼苗生长
    选择均匀发芽的种子,并置于96孔板中,然后转移至清水,直到根长度达到3cm,之后将种子移植到改良的木村B溶液中。水稻幼苗在具有12小时光照(30℃)/12小时黑暗(28℃)光周期和70%湿度的生长室中生长约2周。解决方案每天更换。
  3. 15 硝酸盐摄取测定
    在2周培养后,用改良的木村B溶液预处理水稻幼苗2小时,之后用自来水将水稻根洗涤两次,将水稻幼苗转移到含有5mM L-苏式> N-KNO 3 3小时。
  4. 稻苗收获
    吸收3小时后,用0.1mM CaSO 4洗涤水稻2分钟,以除去N-NO 3 - 反 - 在根表面上,然后单独收获根和芽,并在70℃下干燥至恒重,在纸袋中。干燥的样品在研钵中研磨成细粉,用于随后的测定
  5. 氮吸收计算
    通过同位素比质谱仪分析约0.5mg干燥粉末,并获得 N含量的数据(Brand,1996)。在检测15-硝酸盐吸收活性的同时,测定根(DWR)/3的总植物(TN)/干重(DW)的总量h]应用于计算,即,每单位时间的根每单位重量的摄取量 15 N的总量。全株的量是从芽数和根数的总和得到的。使用茎苗15 N含量(SN)与根茎15 N含量(RN)的比率来表示根到茎的转运活性(值越高,较高的根到茎转运活性)。

代表数据



图1.从底部切下的96孔板,非常适合水稻幼苗的生长。 放入96孔板后,水稻根可以向下生长成溶液,而水稻芽可以沿着每个井向上生长。


图2.在96孔板上生长的10日龄水稻幼苗。 96孔板下面的容器中装满了改良的木村B溶液,通常容器用不透光材料包裹(例如 ,锡箔)以保护稻根免受光照
Table1。 15 N的芽和根的


Table2。计算 15 硝酸盐吸收活性和根到茎的转运活性

笔记

  1. 选择均匀发芽的种子以确保不同品系的相同生长状态
  2. 发芽的种子可以转移到改良的木村B溶液中,直到根长度为约3至5cm
  3. 用改良的木村B溶液预处理水稻幼苗2小时的目的是确保水稻幼苗在 15硝酸盐摄取测定之前恢复到相对正常的生理状态。

食谱

  1. 改良的木村B溶液
    5mM KNO 3
    0.36mM CaCl 2。 0.54mM MgSO 4 0.18mM KH 2 PO 4 sub/
    40μMFeSO 4 -EDTA
    18.8μMH sub 3 BO 3
    13.4μMMnCl 2
    0.32μMCuSO 4
    0.3μMZnSO 4
    0.03μMNa 2 MoO 4 sub。 1.6mM Na 2 SiO 3子 pH 6.0
  2. 5mM 15 N-KNO 3
    如在程序3中提及的,使用5mM的15 N-KNO 3来替代经修饰的木村B溶液中的5mM KNO 3,而其它成分 保持不变。
  3. 0.1mM CaSO 4溶液
    在1L去离子水中溶解0.0172g CaSO 4·2H 2 O [

致谢

水培培养已在Lin等人(2008)和Hu等人(2015), 15硝酸盐摄取中被照射活性和根到茎转运活性测定由Hu等人(2015)引用。这项工作得到了中国科学技术部(2014AA10A602-5,2015CB755702)和中国科学院(XDA08010400)的资助。

参考文献

  1. Brand,W.A。(1996)。 质谱中的高精度同位素比率监测技术 /em> 31:225-235。
  2. Delhon,P.,Gojon,A.,Tillard,P。和Passama,L。(1995)。 NO <3>的日期调节在大豆植物中的摄取I.在昼夜循环中植物中NO 3的升高 - 流入,流出和N利用的变化。 实验植物学杂志 46(291):1585-1594。
  3. LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC LC ,Piao,ZZ,Deng,QY,Deng,K.,Xu,C.,Liang,Y.,Zhang,LH,Li,LG和Chu,CC(2015)。 NRT1.1B的变化有助于水稻亚种之间的硝酸盐使用差异。 Nature Genetics 47(7):834-838。
  4. Lin,SH,Kuo,HF,Canivenc,G.,Lin,CS,Lepetit,M.,Hsu,PK,Tillard,P.,Lin,HL,Wang,YY,Tsai,CB,Gojon, YF(2008)。 拟南芥 NRT1.5硝酸盐转运蛋白的突变导致根部到芽的硝酸盐运输。 植物细胞 20(9):2514-2528
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引用:Liu, Y., Hu, B. and Chu, C. (2016). 15N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice. Bio-protocol 6(16): e1897. DOI: 10.21769/BioProtoc.1897.
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