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Extraction of Ions from Leaf Sections
叶片切片的离子提取   

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Abstract

The concentration of ions in plant cells and tissues is an important factor to determine their functions and conditions. Here, we describe the method to extract ions from leaf sections for measurements with an ion chromatogram. This method is available for not only barley but also other plant species.

Keywords: Ion chromatography(离子色谱法), Barley(大麦), Inorganic ion(无机离子)

Materials and Reagents

  1. Barley seedlings
  2. Milli-Q water
  3. Hydroponic culture solution (see Recipes)

Equipment

  1. 1.5 ml and 2 ml plastic tubes
  2. Scissors and forceps
  3. Sample crusher (Kurabo Industries, model: SH-48 )
  4. 7 mm zirconia beads (Kurabo Industries, model: Z-07 )
  5. Vortex mixer
  6. Dry thermo unit (Taitec, catalog number: DTU-18 )
  7. 0.45 µm filters (EMD Millipore, catalog number: SLLHH04NL )
  8. 1.0 ml syringe
  9. Centrifuge
  10. Ion chromatogram (Thermo Fisher Scientific, Dionex, model: ICS-1500 ) equipped with an ion exchange column (AS-12 for anions and CS-16 for cations)

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. Leaves are sampled using scissors and forceps and weighed.
  3. Samples are cut into small pieces, then put into 2 ml plastic tubes with 7 mm zirconia beads and frozen in liquid nitrogen.
  4. Frozen leaves are crushed to a powder using a sample crusher.
  5. 1 ml distilled water is added to each sample which is then homogenized on a vortex mixer at the maximum speed (e.g. 3,000 rpm) until the mixture becomes uniform.
  6. Samples are centrifuged at 20,400 x g for 10 min at 4 °C. The supernatant is put into a 1.5 ml plastic tube and boiled at 100 °C for 7 min with a dry thermo unit to denature enzymes like phosphatases in Nagai et al. (2013). If samples are heated for longer than 7 min, some metabolites may begin to be degraded.
  7. Samples are cooled on ice and again centrifuged at 20,400 x g for 10 min at 4 °C.
  8. The supernatant is sucked into a 1 ml syringe, and then filtered through a 0.45 µm filter to remove debris.
  9. The filtrates are stored at -20 °C until measurement.
  10. Ion levels in each sample are determined with an ion chromatogram.

Notes

  1. All steps should be performed on ice to avoid degradation of organic ions.
  2. Depending on the purpose of the experiment, any part of the leaf can be used. The extract from at least 5 mg of sample is needed for the ion chromatogram. Sample amounts may vary according to the nutritional status of the plants, but must be enough for the detection by the apparatus.
  3. The size and number of zirconia beads in each plastic tube should be adjusted according to the hardness of the sample. In case of a barley primary leaf (about 100 mg), we used two beads with our sample crasher.
  4. To crush a larger sample, a mortar and pestle is available. In that case, samples are put into a mortar with liquid nitrogen, and ground by a pestle.
  5. For details about the ion chromatography, see Weiss and Weiss (2014).

Recipes

  1. Hydroponic culture solution
    9 mol/m3         KNO3
    6 mol/m3         Ca (NO3)2
    3 mol/m3         MgSO4
    1.5 mol/m3      KH2PO4
    0.125 mol/m3   Fe-EDTA
    Micronutrients: 10 mmol/m3 MnSO4, 1 mmol/m3 CuSO4, 1 mmol/m3 ZnSO4, 30 mmol/m3 H3BO3, 30 µmol/m3 (NH4)6Mo7O24, 0.1 mmol/m3 CoCl2

Acknowledgments

The present protocol was adapted from the following publication: Nagai et al. (2013). 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).

References

  1. 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.
  2. Weiss, J. and Weiss, T. (2004). Handbook of Ion Chromatography, Third, Completely Revised and Enlarged Edition. Wiley-VCH Verlag GmbH.

简介

植物细胞和组织中的离子浓度是确定它们的功能和条件的重要因素。 在这里,我们描述从叶片中提取离子的离子色谱图测量方法。 该方法不仅可用于大麦,而且可用于其它植物物种。

关键字:离子色谱法, 大麦, 无机离子

材料和试剂

  1. 大麦幼苗
  2. Milli-Q水
  3. 水培培养液(见食谱)

设备

  1. 1.5ml和2ml塑料管
  2. 剪刀和镊子
  3. 样品破碎机(Kurabo Industries,型号:SH-48)
  4. 7毫米氧化锆珠(Kurabo Industries,型号:Z-07)
  5. 涡街搅拌机
  6. 干热单元(Taitec,目录号:DTU-18)
  7. 0.45μm过滤器(EMD Millipore,目录号:SLLHH04NL)
  8. 1.0 ml注射器
  9. 离心机
  10. 配有离子交换柱(阴离子的AS-12和阳离子的CS-16)的离子色谱图(Thermo Fisher Scientific,Dionex,型号:ICS-1500)

程序

  1. 大麦植物在潮湿滤纸上发芽2-3天,然后将幼苗在水培培养中生长7-8天。
  2. 叶子用剪刀和镊子取样,称重。
  3. 将样品切成小块,然后放入2ml具有7mm氧化锆珠的塑料管中并在液氮中冷冻
  4. 使用样品破碎机将冷冻的叶子粉碎成粉末。
  5. 将1ml蒸馏水加入到每个样品中,然后以最大速度(例如3000rpm)在涡旋混合器上均化,直到混合物变得均匀。
  6. 将样品在4℃下以20,400×g离心10分钟。将上清液放入1.5ml塑料管中,并用干热单元在100℃下煮沸7分钟,以使Nagai等人(2013)中的酶像磷酸酶变性。如果样品加热超过7分钟,一些代谢物可能开始降解
  7. 将样品在冰上冷却,并在4℃下再次以20,400×g离心10分钟。
  8. 将上清液吸入1ml注射器,然后通过0.45μm过滤器过滤以除去碎屑。
  9. 滤液保存在-20℃,直至测定。
  10. 每个样品中的离子浓度用离子色谱法测定

笔记

  1. 所有步骤都应在冰上进行,以避免有机离子退化
  2. 根据实验的目的,可以使用叶子的任何部分。离子色谱图需要至少5 mg样品的提取物。样品量可能会根据植物的营养状况而有所不同,但必须足以进行设备的检测
  3. 每个塑料管中氧化锆珠的大小和数量应根据样品的硬度进行调整。在大麦原叶(约100毫克)的情况下,我们用我们的样品蛤蜊用了两颗珠子
  4. 为了粉碎较大的样品,可以使用砂浆和杵。在这种情况下,将样品放入带有液氮的砂浆中,并用杵研磨
  5. 有关离子色谱的详细信息,请参阅Weiss和Weiss(2014)。

食谱

  1. 水培培养液
    9 mol / m 3         KNO 3
    6 mol / m 3         Ca(NO 3 2
    3 mol / m 3       < MgSO 4< / sub>
    1.5摩尔/平方米      KH 2 PO 4
    0.125mol / m 3   Fe-EDTA
    微量营养素:10mmol / m 3,MnSO 4,1mmol / m 3,3%以上的CuSO 4,1mmol / m 2 3o 3,3×10 3,3×10 3,3×10 3,3×10 3 μmol/ m 3(NH 4)6 6 Mo 7 O 24,0.1 mmol / m 3 / sup> CoCl 2

致谢

本协议从以下出版物改编:永井等人(2013)。这项工作部分由教育,文化,体育,科技和日本科学促进会(JSPS)的科学研究资助单,JST(日本科技公司)的CREST部分支持, 。

参考文献

  1. 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)。 与大麦初级叶片的组织离子水平相关的木质部渗出物和排液中的离子梯度。 a>植物细胞环境 36(10):1826-1837。
  2. Weiss,J.和Weiss,T。(2004)。 离子色谱手册,第三,完全修订和扩大版。 Wiley-VCH Verlag GmbH。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kurita, Y., Kanno, S., Ohnishi, M. and Mimura, T. (2014). Extraction of Ions from Leaf Sections. Bio-protocol 4(13): e1174. DOI: 10.21769/BioProtoc.1174.
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