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Autoradiography of Pi Distribution in Barley Seedlings
大麦幼苗中磷分布的放射性自显影   

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Abstract

Phosphorus-32 and Phosphorus-33 are radioisotopes of phosphorus. These isotopes are used to trace ionic phosphorus and phosphorus compounds. This protocol is used to follow the movement of inorganic phosphate (PO43-) from a leaf tip to the rest of the plant.

Keywords: Autoradiogram(放射自显影), Imaging plate(成像板), Barley(大麦), Phosphate(磷酸盐), Translocation(易位)

Materials and Reagents

  1. Barley seedlings
  2. Radioisotopes 32P or 33P labeled NaH2PO4 dissolved in water (MP Biomedicals, PerkinElmer or American Radiolabeled Chemicals)
  3. 5 mM CaSO4 solution
  4. Hydroponic culture solution (see Recipes)

Equipment

  1. Cling film
  2. 1.5 ml plastic tubes
  3. 15 ml plastic tubes (1.5 ml tube is fitted by opening a hole in the lid) (Figure 1)
  4. Cotton
  5. Plastic sponge
  6. Imaging plate (FCR Imaging Plate for general purpose) (Fujifilm Corporation) and plate cassette (FCR standard cassette) (Fujifilm Corporation)
  7. Imaging analyzer (GE Healthcare, model: Typhoon 9400 or other Radioisotope imaging analyzers)

Procedure

  1. Barley plants are germinated on moist filter paper for 2-3 days and then seedlings are grown in hydroponic culture for 7-8 days.
  2. Cotton is put in a 1.5 ml tube from which the cap has been removed, then a radioisotope medium consisting of 600 µl of 0.2  mM NaH232PO4 (specific activity 3.7 MBq nmol−1) in 5 mM CaSO4 is added. Cotton is put enough to absorb all 600 µl NaH232PO4, But, do not put too much cotton to keep it moisture.
  3. Into the 15 ml tube, an appropriate amount of incubation medium (5 mM CaSO4) is added. A barley plant sandwiched with sponge is put into the medium, and the barley leaf is manipulated into position against the plastic sponge separated from solution, such that when the smaller tube containing the cotton is mounted into a hole in the cap of the 15 ml tube, the tip of the leaf comes into contact with the cotton soaked in radioactive medium (Figure 1).


    Figure 1. Setup for the radioisotopic labelling of a leaf tip

  4. After an appropriate labelling period (about 2 h) at 25 degrees, the sample is washed with water several times and wrapped with cling film. The sample is then set on an imaging plate and exposed (Figure 2).


    Figure 2. Setup of the radiolabeled sample on an imaging plate

    To avoid contamination of imaging plate with 32P, samples are wrapped by cling film. Wrapped sample is attached to the imaging plate by using binding case to expose.

  5. Examine the imaging plate with an imaging analyzer (Figure 3). 32P concentration is indicated by pseudo-color.


    Figure 3. Autoradiogram of 32P distribution in a barley plant which was radiolabeled from the leaf tip

  6. Following imaging, plants can continue to be incubated in 5 mM CaSO4 solutions under the same light conditions for 1 or 2 d without cap in order to examine the movement of the radioisotope. In Figure 3 it can be seen that NaH232PO4 absorbed at the leaf tip was translocated to the root after one day of incubation.
    Figure 4 shows autoradiograms taken every 2 days following the initial incubation of roots in a Pi-deficient barley plant with an appropriate amount of incubation medium mixed NaH232PO4 (Mimura et al., 1996)


    Figure 4. Autoradiogram of 32P distribution in 10 days old barley plant grown in a Pi-deficient medium, which was radiolabeled from the root. The number put at the leaves in order of development.

Notes

  1. Resolution and clarity of images are strongly dependent on the radioisotope contents of plants and exposure time on the imaging plate.

Recipes

  1. Hydroponic culture solution
    9 mM KNO3
    6 mM Ca (NO3)2
    3  mM MgSO4
    1.5  mM KH2PO4
    0.125 mM Fe-EDTA
    Micronutrients: 10 µM MnSO4, 1 µM CuSO4, 1 µM ZnSO4, 30 µM H3BO3, 30 µM (NH4)6Mo7O24, 0.1  µM CoCl2

Acknowledgments

This protocol was adapted from the following publications, Nagai et al. (2013) and Mimura et al. (1996). This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and Japan Society for the Promotion of Science (JSPS), CREST of JST (Japan Science and Technology Corporation) and in part by Hyogo Science and Technology Association. The authors also thank Yokogawa Analytical Systems Inc. for use of ion chromatography equipment.

References

  1. Mimura, T., Sakano, K. and Shimmen, T. (1996). Studies on the distribution, re‐translocation and homeostasis of inorganic phosphate in barley leaves. Plant Cell Environ 19(3): 311-320.
  2. Nagai, M., Ohnishi, M., Uehara, T., Yamagami, M., Miura, E., Kamakura, M., Kitamura, A., Sakaguchi, S., Sakamoto, W., Shimmen, T., Fukaki, H., Reid, R. J., Furukawa, A. and Mimura, T. (2013). Ion gradients in xylem exudate and guttation fluid related to tissue ion levels along primary leaves of barley. Plant Cell Environ 36(10): 1826-1837.

简介

磷-32和磷-33是磷的放射性同位素。 这些同位素用于追踪离子磷和磷化合物。 该方案用于跟踪从叶尖到植物其余部分的无机磷酸盐(PO 4+)的移动。

关键字:放射自显影, 成像板, 大麦, 磷酸盐, 易位

Materials and Reagents

  1. Barley seedlings
  2. Radioisotopes 32P or 33P labeled NaH2PO4 dissolved in water (MP Biomedicals, PerkinElmer or American Radiolabeled Chemicals)
  3. 5 mM CaSO4 solution
  4. Hydroponic culture solution (see Recipes)

Equipment

  1. Cling film
  2. 1.5 ml plastic tubes
  3. 15 ml plastic tubes (1.5 ml tube is fitted by opening a hole in the lid) (Figure 1)
  4. Cotton
  5. Plastic sponge
  6. Imaging plate (FCR Imaging Plate for general purpose) (Fujifilm Corporation) and plate cassette (FCR standard cassette) (Fujifilm Corporation)
  7. 成像分析仪(GE Healthcare,型号:Typhoon 9400或其他放射性同位素成像分析仪)

程序

  1. 大麦植物在潮湿的滤纸上发芽2-3天,然后将苗在水培培养中生长7-8天。
  2. 将棉放入已经除去盖子的1.5ml管中,然后加入由600μl0.2mM NaH 2 SO 4,PO 4 PO 4组成的放射性同位素培养基,加入5mM CaSO 4中的抗体(比活性3.7MBq nmol -1)。棉花被放到足以吸收所有600μlNaH 2 SO 4,<32> PO 4,但是,不要放太多的棉以保持水分。 br />
  3. 向15ml管中加入适量的培养基(5mM CaSO 4)。将夹在海绵中的大麦植物放入培养基中,并将大麦叶对着从溶液中分离的塑料海绵操纵到位,使得当含有棉花的较小管安装到15ml管的盖中的孔中时,叶片的尖端与浸泡在放射性介质中的棉花接触 (图1)。


    图1.叶尖的放射性同位素标记设置

  4. 在25度适当的标记期(约2小时)后,将样品用水洗涤数次并用保鲜膜包裹。然后将样品置于成像板上并暴露(图2)

    图2.在成像板上设置放射性标记的样品

    为了避免成像板被 32 P污染,样品被保鲜膜包裹。包装的样品通过使用装订盒暴露而连接到成像板
  5. 用成像分析仪检查成像板(图3)。 32 P浓度由伪色指示。


    图3.从叶子放射标记的大麦植物中 3 2 P分布的放射自显影提示

  6. 在成像之后,植物可以在相同光照条件下在5mM CaSO 4溶液中继续孵育1或2天,无盖,以检查放射性同位素的移动。在图3中,可以看出,在孵育一天后,在叶尖吸收的NaH 2+ 32+ PO 4被易位到根。
    图4显示了在缺乏Pi的大麦植物中根的初始温育之后每2天取的放射自显影图,所述大麦植物具有适当量的混合了NaH 2 S 2 PO 4, 4 (Mimura et al。,1996)


    图4. 32 P分布在10天大麦植物中的生长在Pi缺陷培养基中的放射自显影根。按照开发顺序放在叶子上的数字。

笔记

  1. 图像的分辨率和清晰度强烈依赖于植物的放射性同位素含量和成像板上的暴露时间。

食谱

  1. 水培培养液
    9 mM KNO 3
    6mM Ca(NO 3)2 sub。 3mM MgSO 4 1.5mM KH 2 PO 4 4/v/v 0.125mM Fe-EDTA 微量营养素:10μMMnSO 4,1μMCuSO 4,1μMZnSO 4,30μMH 3 BO 3 NH 3,30μM(NH 4)6 Mo 7 SO 24,0.1μM(NH 4) μMCoCl 2

致谢

该方案改编自以下出版物,Nagai等人(2013)和Mimura等人(1996)。这项工作得到了教育,文化,体育,科学技术部科学研究助理和日本科学促进会(JSPS),JST(日本科学技术公司)CREST的部分支持,并且部分由兵库科学技术协会。作者还感谢Yokogawa Analytical Systems Inc.使用离子色谱设备。

参考文献

  1. Mimura,T.,Sakano,K。和Shimmen,T。(1996)。 无机磷酸盐的分布,再移位和体内平衡研究在大麦叶中。 Plant Cell Environ 19(3):311-320
  2. Nagai,M.,Ohnishi,M.,Uehara,T.,Yamagami,M.,Miura,E.,Kamakura,M.,Kitamura,A.,Sakaguchi,S.,Sakamoto,W.,Shimmen, Fukaki,H.,Reid,RJ,Furukawa,A。和Mimura,T。(2013)。 与沿大麦主叶的组织离子水平相关的木质部渗出物和喷射流体中的离子梯度。 a> Plant Cell Environ 36(10):1826-1837。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kanno, S., Kurita, Y., Ohnishi, M. and Mimura, T. (2014). Autoradiography of Pi Distribution in Barley Seedlings. Bio-protocol 4(13): e1175. DOI: 10.21769/BioProtoc.1175.
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