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Arabidopsis thaliana Root Hair Cell Cytoplasmic pH (pHc) Imaging
拟南芥根毛细胞质pH(pHc)的成像   

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Abstract

For monitoring the cellular pH in plants, engineered green fluorescent protein (GFP) has been used to indicate cellular pH. Pt-GFP is a pH-reporter protein which has been used ratiometrically in the double excitation mode, according to the fluorescence properties described for Pt-GFP (Schulte et al., 2006), and depending on our previous experiments, we present here a little and efficient protocol for monitoring the cytosolic pH value. In this protocol, root hair cellular pH was monitored in the PtGFP reporter lines. Cellular pH can be obtained according to the calibration curve which was performed in situ by using a series pH buffer.

Keywords: Cytoplasmic pH(胞质pH值), Root hair(根头发), GFP(GFP)

Materials and Reagents

  1. Stable Pt-GFP transgenic WT line N9561 (background: Col-0) was obtained from the Nottingham Arabidopsis Stock Centre http://arabidopsis.info/StockInfo?NASC_id=9561).
    Note: Pt-GFP T-DNA insertion mutant homozygous were obtained by crossing the T-DNA insertion mutant (what you interested, here meant the mutant with root hair growth problem) with the N9561. And we obtained the T-DNA insertion homozygous first by PCR (methods in http://signal.salk.edu/cgi-bin/tdnaexpress) in the F2 progeny, and we checked the fluorescence for Pt-GFP. Homozygous lines with fluorescence will be used for later analysis.
  2. Digitonin (Sigma-Aldrich, catalog number: 11024-24-1 )
    Note: Risk assessment: toxic. Digitonin is toxic by inhalation, contact with skin, wear protective gloves and clothing, wear respiratory protection, when handling this chemical.
  3. 2-(N-Morpholino) ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: 145224-94-8 )
  4. N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: 7365-45-9 )
  5. Murashige and Skoog (MS) medium (Phyto Tech, catalog number: M519 )
  6. pH buffer solutions (see Recipes)

Equipment

  1. Inverted Carl Zeiss LSM 710 confocal microscope
  2. Plant growth chamber
  3. 2 ml tubes (Eppendorf tubes)
  4. Forceps
  5. Microscope slides
  6. Microscope cover slips

Software

  1. Microsoft Excel

Procedure

  1. Surface-sterilized Arabidopsis seeds were sowed on 0.5 x MS medium and kept for 3 d at 4 °C in the dark. The plates were then transferred to the growth chamber at 22 °C with a 16-h light/8-h dark photoperiod and grown vertically. Pt–GFP Root hairs of 4-5-d-old seedlings were used to monitor intracellular pH. Seedlings were carefully placed on the slide with 2-3 drops of liquid growth medium (the same as the medium seedling were grown on). Root hair GFP fluorescence was monitored under the confocal microscope (exciters: 405 nm and 488 nm; emitter: 525 nm); the F405-to-F488 ratio was used as a measure for pH, and a pseudocolor-coded 405 nm/488 nm ratio image can be obtained.
  2. The pH titrations were performed in situ as previously reported (Moseyko and Feldman, 2001). For making the calibration curve, a series of pH buffer solutions (5, 5.5, 6, 6.5, 7, 7.5 and 8; for preparation, see Recipes) were used, 4-5-d-old seedlings were incubated in the buffer solution and were stabilized for about 10 min to equilibrate the external pH buffer solutions and the pHc. Fluorescence was made as procedure 1 to obtain F405-to-F488 ratio for different pH buffer solution. For each, at least 5 repeats should be made.
  3. After the in situ titration, an equation (R2 near 0.999) will be reached for conversion the fluorescence ratio to the pH value by using the Microsoft Excel. pH levels of the root hair obtained in step 1 were then calibrated by the equation.
  4. Waste disposal
    Offer surplus and non-recyclable solutions with digitonin to a licensed disposal company. Contact a licensed professional waste disposal service to dispose of this material.

Representative data



Figure 1. pHc in root hairs of Pt-GFP plants. Bars = 10 µm

Recipes

  1. pH buffer solutions
    0.5 x Murashige and Skoog basal salt mix (MS) salts
    0.005% digitonin
    50 mM MES/NaOH (for adjusting pH to 5, 5.5, 6, 6.5) or 20 mM HEPES/NaOH (for pH to 7, 7.5, 8)

Acknowledgments

This work was supported by the National Key Basic Special Funds (2012CB1143001).

References

  1. Moseyko, N. and Feldman, L. J. (2001). Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana. Plant Cell Environ 24(5): 557-563.
  2. Schulte, A., Bottcher, M., Lorenzen, I. and Plieth, C. (2006). A novel pH indicator for expression in plants. Plant Methods 2:7.

简介

为了监测植物中的细胞pH,使用工程绿色荧光蛋白(GFP)来指示细胞pH。 根据Pt-GFP(Schulte等人,2006)所述的荧光性质,Pt-GFP是pH报道蛋白,其已经在双激发模式中按比例计量使用,并且取决于我们 以前的实验,我们在这里提出一个有效的协议,用于监测胞浆pH值。 在该协议中,在PtGFP报告系中监测根毛细胞pH。 可以根据通过使用系列pH缓冲液原位进行的校准曲线获得细胞pH。

关键字:胞质pH值, 根头发, GFP

材料和试剂

  1. 稳定的Pt-GFP转基因野生型系N9561(背景:Col-0)得自诺丁汉拟南芥库存中心(Nottingham Arabidopsis Stock Center) NASC_id = 9561"> http://arabidopsis.info/StockInfo?NASC_id=9561 )。
    注意:Pt-GFP T-DNA插入突变体纯合子通过将T-DNA插入突变体(您感兴趣,这里指具有根毛生长问题的突变体)与N9561杂交获得。我们首先通过PCR获得T-DNA插入纯合子( http ://signal.salk.edu/cgi-bin/tdnaexpress ),我们检查了Pt-GFP的荧光。具有荧光的纯合系将用于以后的分析。
  2. digitonin(Sigma-Aldrich,目录号:11024-24-1)
    注意:风险评估:有毒。在使用此化学品时,吸入有毒,吸入皮肤,戴防护手套和衣服,戴呼吸防护用品。
  3. 2-(N-吗啉代)乙磺酸(MES)(Sigma-Aldrich,目录号:145224-94-8)
  4. N-2-羟乙基哌嗪-N'-2-乙磺酸(HEPES)(Sigma-Aldrich,目录号:7365-45-9)
  5. Murashige和Skoog(MS)培养基(Phyto Tech,目录号:M519)
  6. pH缓冲溶液(参见配方)

设备

  1. 倒立的Carl Zeiss LSM 710共焦显微镜
  2. 植物生长室
  3. 2ml管(Eppendorf管)
  4. 镊子
  5. 显微镜载玻片
  6. 显微镜盖玻片

软件

  1. Microsoft Excel

程序

  1. 将表面灭菌的拟南芥种子播种在0.5×MS培养基上,并在4℃在黑暗中保持3天。然后将板在22℃下用16小时光/8小时黑暗光周期转移到生长室中并垂直生长。 Pt-GFP 4-5-d龄幼苗的根毛用于监测细胞内pH。将幼苗小心地置于载有2-3滴液体生长培养基(与培养基幼苗生长相同)上的载玻片上。在共焦显微镜下(激发子:405nm和488nm;发射器:525nm)监测根毛GFP荧光;使用F405至F488比率作为pH的测量,并且可以获得伪彩色编码的405nm/488nm比的图像。
  2. 如先前报道的(Moseyko和Feldman,2001)原位进行pH滴定。为了制备校准曲线,使用一系列pH缓冲溶液(5,5.5,6,6.5,7,7.5和8;用于制备,参见Recipes),将4-5天龄的幼苗在缓冲溶液并稳定约10分钟以平衡外部pH缓冲溶液和pHc。如程序1进行荧光,以获得不同pH缓冲溶液的F405至F488比例。对于每个,至少应重复5次。
  3. 在原位滴定之后,将使用Microsoft Excel将荧光比转换为pH值的方程式(R近似为0.999)。步骤中获得的根毛的pH水平 1然后通过公式校准
  4. 废物处理
    向经许可的处理公司提供含有洋地黄皂苷的剩余和不可回收解决方案。 联系专业废物处理服务处理此材料。

代表数据



图1. Pt-GFP植物根毛中的pHc。 棒=10μm

食谱

  1. pH缓冲溶液
    0.5×Murashige和Skoog基础盐混合物(MS)盐
    0.005%的洋地黄素
    50mM MES/NaOH(用于调节pH至5,5.5,6,6.5)或20mM HEPES/NaOH(pH至7,7.5,8)

致谢

这项工作得到国家关键基本特别基金(2012CB1143001)的支持。

参考文献

  1. Moseyko,N。和Feldman,L.J。(2001)。 pH敏感性绿色荧光蛋白在拟南芥中的表达 植物细胞 Environ 24(5):557-563。
  2. Schulte,A.,Bottcher,M.,Lorenzen,I。和Plieth,C。(2006)。 用于在植物中表达的新型pH指示剂植物方法 2:7。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bai, L., Zhou, Y., Wang, P. and Song, C. (2015). Arabidopsis thaliana Root Hair Cell Cytoplasmic pH (pHc) Imaging . Bio-protocol 5(7): e1438. DOI: 10.21769/BioProtoc.1438.
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