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Measurement of Potassium Content in Arabidopsis
拟南芥中钾含量的测量

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Abstract

Potassium is an essential element in plant growth and has an important role in regulating cell water potential and turgor in osmotic regulation. Potassium content in plants is high compared to trace elements, however, it is difficult to measure a relatively small change of potassium content in the large total. Here, we describe a procedure for measuring potassium in Arabidopsis that is easy to handle in preparative scale and avoids contamination.

Materials and Reagents

  1. Arabidopsis seedlings
  2. Milli-Q water
  3. Nitric acid (analytical grade for heavy metals) (Wako Pure Chemical Industries, catalog number: 140-04016 )
  4. Potassium standard solution (1,000 ppm) (Wako Pure Chemical Industries, catalog number: 165-17471 )
  5. Contaminon L (Wako Pure Chemical Industries, catalog number: 035-09311 )
  6. Murashige and Skoog salt (Wako Pure Chemical Industries, catalog number: 392-00591 )
  7. 2-(N-morpholino)ethanesulfonic acid (MES, Wako Pure Chemical Industries, catalog number: 345-01625 )
  8. Gamborg’s Vitamin Solution (Sigma-aldrich, catalog number: G1019 )
  9. Difco Bacto agar (Becton, Dickinson and Company, catalog number: 214010 )
  10. Germination media (GM)-agar (see Recipes)

Equipment

  1. Plates
  2. Centrifugation tubes
  3. A paper or aluminum foil
  4. Atomic absorption spectrometer (AAS) or Inductively-coupled plasma optical emission spectrometer (ICP-OES)
  5. Stainless-steel vessel (HU-25, o.d. = 55 mm x 104 mm) (SAN-AI science)
  6. Polytetrafluoroethylene (PTFE) vessel (o.d. = 28 mm x 40 mm) (SAN-AI science)
  7. Tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA) cup (o.d. = 22 mm x 32 mm, 7 ml) (Sanplatec, catalog number: 0225R )
  8. Volumetric flask (100 ml)
  9. Ceramic scissors and tweezers (AS ONE Corporation, catalog number: 8-203-23 , 7-166-11 )

Procedure

  1. Arabidopsis seedlings are germinated for 10 days on GM agar and then cultivated hydroponically with milliQ H2O in plates for 5 days.
  2. Plants are dissected into shoots and roots by cutting the bottom of the shoots with ceramic scissors and tweezers.
  3. Samples are dried in disposable centrifugation tubes (50 ml) at 70 °C for 12 h. A paper or aluminum foil is placed over the samples during the drying process.
  4. Dried samples (between 15 and 20 plants, corresponding to between 5 and 10 mg as dry weight) are placed into PFA cups with ceramic tweezers and weighed.
  5. The PFA cups are placed in PTFE vessels, and nitric acid (1 ml) is added to the PTFE vessels outside the PFA cups. To avoid contamination of impurities in nitric acid, samples are decomposed by nitric acid vapor (not nitric acid liquid).
  6. The PTFE vessels are set in stainless-steel vessels (HU-25) and heated at 150 °C for 10 h.
  7. The decomposed samples in the PFA cups are diluted with 0.1 M nitric acid and poured into a volumetric flask (100 ml). The sample solution is filled up with 0.1 M nitric acid and subjected to analysis.
  8. Potassium content in plants is measured with an atomic absorption spectrometer in emission mode. Standard solutions at concentrations of 0, 1, 2, 4, 8 ppm are prepared from a 1,000 ppm reference solution diluted with 0.1 M nitric acid (It also can be measured by ICP-OES).

Notes

  1. To avoid potassium contamination from hands, use rubber gloves during sample treatments.
  2. Take care when handling dried samples, which may be attached to vessel walls or scattered due to static electricity.
  3. Dried samples are very hygroscopic. It is necessary to weigh them quickly for an accurate content calculation.
  4. Contaminon L is used for heavy metal cleaning. Glass vessels are immersed in Contaminon L overnight and rinsed off with MilliQ water before use.

Recipes

  1. GM (germination media)-agar
    1x Murashige and Skoog salt
    3% sucrose
    1x Gamborg’s Vitamin solution
    0.05% 2-(N-morpholino)ethanesulfonic acid (MES)
    0.8% Difco Bacto agar
    Adjusted to pH 5.7 with 1 M KOH

Acknowledgments

This protocol is based on the procedure described by Kojima and Iida (1986).

References

  1. KOJIMA, I. and IIDA, C. (1986). Phase digestion of botanical samples polytetrafluoroethylene bomb. Anal Sci 2: 567. 
  2. Osakabe, Y., Arinaga, N., Umezawa, T., Katsura, S., Nagamachi, K., Tanaka, H., Ohiraki, H., Yamada, K., Seo, S. U., Abo, M., Yoshimura, E., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2013). Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis. Plant Cell 25(2): 609-624. 

简介

钾是植物生长中的必需元素,并且在调节细胞水势和渗透调节中的膨胀中具有重要作用。 与微量元素相比,植物中的钾含量高,然而,难以在大的总量中测量钾含量的相对小的变化。 在这里,我们描述了在拟南芥中测量钾的程序,其易于在制备规模中处理并避免污染。

材料和试剂

  1. 拟南芥苗
  2. Milli-Q水
  3. 硝酸(重金属的分析纯)(Wako Pure Chemical Industries,目录号:140-04016)
  4. 钾标准溶液(1000ppm)(Wako Pure Chemical Industries,目录号:165-17471)
  5. Contaminon L(Wako Pure Chemical Industries,目录号:035-09311)
  6. Murashige和Skoog盐(Wako Pure Chemical Industries,目录号:392-00591)
  7. 2-(N-吗啉代)乙磺酸(MES,Wako Pure Chemical Industries,目录号:345-01625)
  8. Gamborg's维生素溶液(Sigma-aldrich,目录号:G1019)
  9. Difco Bacto琼脂(Becton,Dickinson and Company,目录号:214010)
  10. 萌发培养基(GM) - 琼脂(见配方)

设备


  1. 离心管
  2. 纸张或铝箔
  3. 原子吸收光谱仪(AAS)或电感耦合等离子体发射光谱仪(ICP-OES)
  4. 不锈钢容器(HU-25,o.d. = 55mm×104mm)(SAN-AI科学)
  5. 聚四氟乙烯(PTFE)容器(o.d. = 28mm×40mm)(SAN-AI科学)
  6. 四氟乙烯 - 全氟烷基乙烯基醚共聚物(PFA)杯(o.d. = 22mm×32mm,7ml)(Sanplatec,目录号:0225R)
  7. 容量瓶(100ml)
  8. 陶瓷剪刀和镊子(AS ONE公司,目录号:8-203-23,7-166-11)

程序

  1. 拟南芥幼苗在GM琼脂上萌发10天,然后用milliQ H 2 O在板中水培5天。
  2. 通过用陶瓷剪刀和镊子切割芽的底部,将植物解剖成枝和根。
  3. 将样品在一次性离心管(50ml)中在70℃下干燥12小时。在干燥过程中,将纸或铝箔放在样品上
  4. 将干燥的样品(15至20株植物,相当于5至10mg干重)放入带有陶瓷镊子的PFA杯中并称重。
  5. 将PFA杯放置在PTFE容器中,并将硝酸(1ml)加入到PFA杯外部的PTFE容器中。为了避免硝酸中的杂质污染,样品通过硝酸蒸气(不是硝酸液体)分解
  6. 将PTFE容器置于不锈钢容器(HU-25)中,并在150℃下加热10小时
  7. 将PFA杯中的分解样品用0.1M硝酸稀释并倒入容量瓶(100ml)中。样品溶液用0.1M硝酸填充并进行分析
  8. 在发射模式下用原子吸收光谱仪测量植物中的钾含量。从用0.1稀释的1,000ppm参考溶液制备浓度为0,1,2,4,8ppm的标准溶液 M硝酸(也可以通过ICP-OES测量)

笔记

  1. 为避免手中的钾污染,在样品处理期间使用橡胶手套
  2. 处理干燥样品时要小心,样品可能附着在容器壁上或由于静电而散落
  3. 干燥样品非常吸湿。 为了准确计算内容,必须快速称重它们。
  4. Contaminon L用于重金属清洗。 将玻璃容器浸入Contaminon L过夜并在使用前用MilliQ水冲洗

食谱

  1. GM(发芽培养基)-agar
    1x Murashige和Skoog盐
    3%蔗糖 1×Gamborg的维生素溶液
    0.05%2-(N-吗啉代)乙磺酸(MES) 0.8%Difco Bacto琼脂 用1M KOH调节至pH 5.7

致谢

该协议基于Kojima和Iida(1986)描述的程序。

参考文献

  1. KOJIMA,I。和IIDA,C。(1986)。 植物样品聚四氟乙烯炸弹相消解。 Anal Sci 2:567. 
  2. Osakabe,Y.,Arinaga,N.,Umezawa,T.,Katsura,S.,Nagamachi,K.,Tanaka,H.,Ohiraki,H.,Yamada,K.,Seo,SU,Abo,M.,Yoshimura ,E。,Shinozaki,K。和Yamaguchi-Shinozaki,K。(2013)。 拟南芥中钾转运蛋白控制的渗透胁迫反应和植物生长。/a> 植物细胞 25(2):609-624。
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引用:Abo, M., Osakabe, Y., Y-Shinozaki, K. and Yoshimura, E. (2013). Measurement of Potassium Content in Arabidopsis. Bio-protocol 3(23): e990. DOI: 10.21769/BioProtoc.990.
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