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Measurement of 33P-PO4 Absorption Capacity and Root-to-leaf Transfer in Arabidopsis
拟南芥吸收33P-PO4的能力和其在根茎中转移的测定   

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Abstract

This method allows quantification of phosphate absorption capacity by Arabidopsis roots using a very simple equipment and it can be scaled up or down.

Keywords: Phosphate absorption(磷的吸收), Phosphate root-to-leaf transfer(磷酸根到叶转移), Arabidopsis(拟南芥)

Materials and Reagents

  1. Plastic 12-well plates (Denmark, Nunc)
    Note: One for the absorption step, one for the desorption step for each treatment (plant type or culture condition).
  2. Plastic 20 ml vials for radioactivity measurement (Ratiolab GmbH, Dreieich)
    Note: You will need one vial per plant, or 2 vials per plant if you want to quantify 33P in both roots and leaves. The vials should be numbered from 1 to N before you start the experiment. They also should be placed in order in appropriate racks (PerkinElmer) adapted to the beta counter.
  3. Tips
  4. Young in vitro plantlets
  5. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
  6. CaCl2 (Sigma-Aldrich)
  7. KH2PO4 (Sigma-Aldrich)
  8. 33P-PO4 5 mCi/ml (40-158 Ci/mg, 1.48-5.84 TBq/mg, >99% isotopically pure, less than 0.5 μM Pi) (PerkinElmer)
  9. Scintillation cocktail (PerkinElmer, Ultima GoldTM)
  10. MgSO4
  11. NH4NO3
  12. KNO3
  13. NaH2PO4
  14. KI
  15. FeCl2
  16. MnSO4
  17. ZnSO4
  18. CuSO4
  19. CoCl2
  20. Na2MoO4
  21. Thiamine
  22. Pyridoxine
  23. Nicotinic acid
  24. Inositol
  25. Sucrose
  26. Agar
  27. MS/10 medium (see Recipes)
  28. Stock solution (see Recipes)
  29. Incubation medium (see Recipes)
  30. Desorption medium (see Recipes)

Equipment

  1. Experiments should be performed on a bench or under a hood illuminated with white light (150 -180 μE m-2 s-1) during the incubation step
  2. Liquid scintillation counter (PerkinElmer, Packard Instrument Company, model: TRI-CARB )
  3. Ice-containing large boxes for the desorption step (all 12-well plates will be placed horizontally on ice for 2 h)
  4. Tweezers for handling the plantlets
  5. If necessary, a razor to separate roots and aerial parts
  6. Micropipets
  7. Shield for protection against radiations (plexiglass)
  8. Scanner or camera (Epson America, model: Perfection V850Pro or Canon, model: Powershot SX130 ), respectively but other devices from other manufacturers could suit perfectly

Software

  1. ImageJ version 1.46r with NeuronJ plugin (http://imagej.nih.gov/ij)

Procedure

  1. Preparation of plant materials
    9 to 11 day-old plantlets are convenient for the experiment.
    1. When plating, space the seeds in order to maintain root systems independent for every single plantlet; this will help when measuring the length of root system (see below). 6 to 10 seeds are sown per plate depending on the growth medium (respectively with high or low Pi, see below). 2 to 3 plates will be necessary per plant type (genotype or treatment depending on the experiment).
    2. Plants are grown vertically in 12 x 12 cm Petri plates in modified MS/10 medium.
    3. Number the plants on each Petri plate, in order to identify each root system individually.
    4. 10 to 12 replicates per treatment or genotype will be necessary plus 5 to 10 plants for blanks.
    5. Scan or photograph of the plates (in black and white and jpg format). Use graph paper as scale bar for measurement of the root length. Scan should be 300 dpi or photos should be about 1,000 x 800 pixels (this is recommended by ImageJ).
    6. Root length measurement and calculation: We use ImageJ version 1.46r with NeuronJ plugin (http://imagej.nih.gov/ij). Figure 1 shows a photo of a plate, and how root length is measured with imageJ. The scale (1 cm using the graph paper) is measured (Figure 1A) then with NeuronJ plugin the primary and lateral roots are traced (pink trace, Figure 1B). Values (in cm) are transferred in an Excel file. Total root length is calculated by adding primary and lateral root lengths (see detailed protocol in Figure 1 legend).


      Figure 1. Root length measurement. A. Set of the scale: with ImageJ, trace a 1 cm scale on the graph paper (1, 2) then ‘set scale’ (3). This gives pixels/cm (4). B. Measurement of the root length: with NeuronJ plugin, load the image file (5) then set scale (as defined in A). Add tracings (6) by drawing the along the roots (pink trace, 7) then measure tracings (RUN, 8). Results of root length in cm (9) can be copied to an excel file. A and B are screenshots of the process.

  2. Incubation
    1. This step should be performed behind a shield for protection against radiations.
    2. Before starting a series of experiments, you must check that Pi absorption is linear in your conditions (plant specificity, temperature, light). To do that, a time course is performed between 30 min and 2 h following the protocol as described below. In our conditions, Pi absorption was always linear.
    3. Prepare12-well plates, one for each plant treatment. Number the wells: 1 to 12.
    4. Add 4 ml of incubation medium per well. Place 1 plant per well with tweezers (Figure 2), the roots should be immersed and the leaves outside the well. To avoid excessive dehydration of the young plantlets during the incubation, carefully put a cover on the plate avoiding the immersion of the leaves in the solution (Figure 2B). Incubate during 2 h at room temperature (22-24 °C) under white light (150-180 μE m-2 s-1). Discard plants that are fully immersed in the liquid, if any.


      Figure 2. Incubation experiment. A. Photo of a 12-well plate with plants numbered 1 to 12 in the incubation medium. The cover plate has been removed for clarity. Please note that rosettes are out of the medium and roots are fully immersed. B. Photo of the plate and the cover (side view) showing how the cover is placed onto the 12-well plate.

    5. In order to evaluate Pi adsorption on the root (mechanical or chemical adsorption can occur on cells but in that case, Pi does not enter inside the root cells), blank samples are treated as follows: The plant root is dipped in the incubation medium for 2 sec (use tweezers) and directly transferred in the desorption medium for 2 h (see below for the desorption procedure).

  3. Desorption (see Video 1)

    Video 1. Desorption

    1. Prepare new 12-well plates, with 3 ml of cold desorption medium per well. Place the plates on ice.
    2. Rinse the incubated plants in water (1-2 sec).
    3. With the help of tweezers, transfer the plants (in the same order you put them in the incubation solution, 1 plant/well) with both leaves and roots immersed in the desorption medium for 2 h on ice.
    4. Transfer each plant in a counting vial (use tweezers). Alternatively, separate the rosette and roots with a sharp razor and put each part of the plant in a separate vial. It is convenient to put the vials in the counting racks as soon as you harvest the plants (in order to avoid mismatches).

  4. Dry the plants in an oven at 50 °C overnight

  5. Radioactivity measurement
    1. Under a chemical hood, add 2 ml of scintillation cocktail and tight a top on each counting vial. Then place the vials in racks adapted to the counter.
    2. With a beta counter, count 33P in each sample (Cs) and in blanks (Cb is a mean of the blank replicates) and also in the incubation medium (C10, 10 μl of incubation medium are placed in a counting vial and 2 ml scintillation cocktail are added). In the scintillation cocktail, beta radiations are transformed in photons that are detected by the beta counter. The measure is in cpm (count per min).
    3. Data analysis:
      The amount of PO4 absorbed per hour per root cm (V in nmol/h/cm) is calculated as follows:
      V = (Cs-Cb) * (S *10-3)* B / (T * Lroot * C10)
      Cs: Radioactivity in the sample (cpm)
      Cb: Mean value of radioactivity in the blanks (cpm)
      S: Pi concentration in the incubation medium (S=50 μM Pi).
      S*10-3=0.05: Pi content (nmol/μl) in the incubation medium (S=50 μM).
      C10: Radioactivity in 10 μl of the incubation solution (cpm). C10/(S*10-2) is the specific activity of 33P in the incubation medium
      B=10: Volume of the incubation solution for measurement of radioactivity before incubation of the plants (10 μl is convenient).
      T=2: Incubation is 2 h
      Lroot: Root length (cm)
      In addition, using the same equation as above, but omitting Lroot, you can calculate Pi absorption per plant (Cs corresponds to 33P in the whole plant), per rosette tissue or root tissue (Cs corresponds to 33P in the leaves or the root system respectively).
      It is also possible to calculate the ratio of 33P (Cs in leaves in cpm) measured in the leaves compared to total 33P absorbed by the whole plant [Cs in leaves + Cs in roots (cpm)].

Representative data

Pi uptake per hour and per root cm for plants grown on low or high Pi (Table 1). Two genotypes are presented (WT and mutant) in order to show the range of variation of Pi uptake between plants and the distribution of Pi in the plant.

Table 1. Pi uptake and distribution in WT and mutant plantlets grown on low or high Pi. Pi uptake capacity is expressed as nmol Pi per hour per root cm (mean±SD). Pi distribution is calculated from the ratio of 33P in leaves compared to 33P in the whole plant.

Recipes

  1. MS/10 medium
    10x time diluted Murashige and Skoog medium containing:
    0.15 mM MgSO4
    2.1 mM NH4NO3
    1.9 mM KNO3
    0.5 (high Pi) or 0.005 (low Pi) mM NaH2PO4
    0.34 mM CaCl2
    0.5 μM KI
    10 μM FeCl2
    10 μM H3BO3
    10 μM MnSO4
    3 μM ZnSO4
    0.1 μM CuSO4
    0.1 μM CoCl2
    1 μM Na2MoO4
    5.9 μM thiamine
    4.9 μM pyridoxine
    8.1 μM nicotinic acid
    55 μM inositol
    3.4 mM MES
    0.5% sucrose and 0.8% agar at pH 5.7
  2. Stock solution
    0.1 mM CaCl2 in 5 mM MES, adjusted at pH 5.5
    For 1 L (water): 0.976 g MES + 0.0147 g CaCl2, adjust pH to 5.5 with 10 N NaOH
  3. 1 M KH2PO4
    For 1 L (water): 136 g KH2PO4
  4. Incubation medium (4 ml/plant are necessary)
    50 µM KH2PO4 in stock solution: 50 µl 1 M KH2PO4 in 1 L stock solution
    Add 5,550 Bq 33P/ml (0.15 µCi 33P/ml)
  5. Desorption medium (3 ml/plant are necessary)
    1 mM KH2PO4 in stock solution: 1 ml 1 M KH2PO4 in 1 L stock solution

Acknowledgments

This protocol was adapted from the previously published studies: Narang et al. (2000) and Misson et al. (2004) and it was performed by Ayadi et al. (2015). This work was supported by the Commissariat à l’Energie Atomique et aux Energies Alternatives.

References

  1. Ayadi, A., David, P., Arrighi, J. F., Chiarenza, S., Thibaud, M. C., Nussaume, L. and Marin, E. (2015). Reducing the genetic redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 transporters to study phosphate uptake and signaling. Plant Physiol 167(4): 1511-1526.
  2. Misson, J., Thibaud, M. C., Bechtold, N., Raghothama, K. and Nussaume, L. (2004). Transcriptional regulation and functional properties of Arabidopsis Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants. Plant Mol Biol 55(5): 727-741.
  3. Narang, R. A., Bruene, A. and Altmann, T. (2000). Analysis of phosphate acquisition efficiency in different Arabidopsis accessions. Plant Physiol 124(4): 1786-1799.

简介

该方法允许使用非常简单的设备通过拟南芥根定量磷酸盐吸收能力,并且其可以按比例放大或缩小。

关键字:磷的吸收, 磷酸根到叶转移, 拟南芥

材料和试剂

  1. 塑料12孔板(丹麦,Nunc)
    注意:一个用于吸收步骤,一个用于每种处理(植物类型或培养条件)的解吸步骤。
  2. 塑料20ml用于放射性测量的小瓶(Ratiolab GmbH,Dreieich)
    注意:如果要量化


    P,每个植物需要一个小瓶 ?在根和叶子。小瓶应从1到N编号 在开始实验之前。他们也应该按顺序放置 适合于β计数器的适当机架(PerkinElmer)。
  3. 提示
  4. 年轻体外苗种
  5. MES水合物(Sigma-Aldrich,目录号:M8250)
  6. CaCl 2(Sigma-Aldrich)
  7. (Sigma-Aldrich)
  8. p-PO 4 5mCi/ml(40-158Ci/mg,1.48-5.84TBq/mg,> 99%同位素纯,小于0.5μMPi )(PerkinElmer)
  9. 闪烁混合物(PerkinElmer,Ultima Gold TM
  10. MgSO 4 4 /
  11. NH 4 3
  12. KNO 3
  13. NaH 2 PO 4 sub
  14. KI
  15. FeCl <2>
  16. MnSO 4
  17. ZnSO 4
  18. CuSO 4
  19. CoCl <2>
  20. Na MoO 4
  21. 硫胺素
  22. 吡哆醇
  23. 烟酸
  24. 肌醇
  25. 蔗糖
  26. Agar
  27. MS/10介质(见配方)
  28. 库存解决方案(参见配方)
  29. 培养基(见配方)
  30. 解吸介质(参见配方)

设备

  1. 实验应当在工作台上或在通过白光照射的罩下(150-180μE m <-2> -1 )进行。孵育步骤
  2. 液体闪烁计数器(PerkinElmer,Packard Instrument Company,型号:TRI-CARB)
  3. 用于解吸步骤的含冰大盒子(所有12孔板将在冰上水平放置2小时)
  4. 用于处理苗的镊子
  5. 如有必要,用剃须刀分离根部和空中部件
  6. 微信
  7. 防护辐射(有机玻璃)的盾牌
  8. 扫描器 ?或相机(Epson America,型号:Perfection V850Pro或Canon,型号: Powershot SX130),但其他设备从其他 制造商可以完美适合

软件

  1. ImageJ版本1.46r与NeuronJ插件( http://imagej.nih.gov/ij

程序

  1. 植物材料的制备
    9到11天的小植物便于实验
    1. 当电镀时,将种子放在空间中以保持根系 独立于每个单株;这将有助于测量时 ?根系长度(见下文)。每板播种6至10粒种子 取决于生长培养基(分别具有高或低的Pi,参见 下面)。每种植物类型需要2到3个平板(基因型或 治疗取决于实验)。
    2. 植物在改良的MS/10培养基中在12×12cm培养皿中垂直生长。
    3. 对每个培养皿上的植物进行编号,以便单独识别每个根系
    4. 每次处理需要10至12个重复,或者加入5至10个空白的植物。
    5. 扫描或照片的板(在黑色和白色和jpg 格式)。使用方格纸作为测量根的比例尺 长度。扫描应为300 dpi,照片应为约1,000 x 800 像素(这是ImageJ推荐的)。
    6. 根长度测量 和计算:我们使用ImageJ版本1.46r与NeuronJ插件 ( http://imagej.nih.gov/ij )。图1显示了一个板的照片,以及如何 根长度用imageJ测量。刻度(1厘米使用图表 纸)(图1A),然后用NeuronJ插件主要和 跟踪侧根(粉红色痕迹,图1B)。值(以cm为单位) 在Excel文件中传输。总根长度通过加法计算 初级和侧根长度(参见图1中的详细方案 图例)。


      图1.根长度测量。A.比例尺设置: 与ImageJ,在方格纸(1,2)上跟踪1厘米的刻度,然后设置 规模'(3)。这给出像素/cm(4)。 B.根长的测量: ?使用NeuronJ插件,加载图像文件(5),然后设置缩放(如定义 ?在一个)。通过沿着根(粉红色痕迹,7)绘制追踪(6) 然后测量描迹(RUN,8)。根长度(cm)(9)的结果可以是 复制到excel文件。 A和B是过程的截图。

  2. 孵化
    1. 此步骤应在屏蔽后面进行,以防止辐射
    2. 在开始一系列实验之前,您必须检查Pi 吸收在您的条件(植物特异性,温度, ?光)。为此,在30分钟和2小时之间进行时间进程 遵循如下所述的方案。在我们的条件下,Pi 吸收总是线性的
    3. 准备12孔板,每个植物处理一个。编号孔:1到12.
    4. 每孔加入4ml培养基。每孔放置1株植物 镊子(图2),根部应浸入和叶子外面 ?井。避免年轻苗期过度脱水 孵化,仔细把盖子盖在盘子上避免 将叶子浸入溶液中(图2B)。孵育2小时 ?在室温(22-24℃)下,在白光下(150-180μE m <-200s -1 )。 丢弃完全浸入液体中的植物,如果有的话

      图 ?2.孵育实验。A.具有植物的12孔板的照片 在孵育培养基中编号1至12。盖板已经 删除为了清楚。请注意,玫瑰花是在媒介和 ?根完全浸没。 B.照片的板和盖(侧面 视图),显示了如何将盖子放置在12孔板上
    5. 为了评估Pi在根上的吸附(机械或 化学吸附可以发生在细胞上,但在这种情况下,Pi不会 进入根细胞内),空白样品处理如下: 将植物根浸泡在培养基中2秒(使用镊子) 并直接在解吸培养基中转移2小时(见下文 ?解吸过程)。

  3. 解吸(见视频1)

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    1. 准备新的12孔板,每孔3毫升冷的解吸培养基。将板放在冰上。
    2. 在水中漂洗孵育的植物(1-2秒)
    3. 在镊子的帮助下,转移植物(以相同的顺序 你把他们在孵化溶液,1植物/井)与两片叶子 并将根在冰上浸泡在解吸介质中2小时
    4.  将每个植物转移到计数瓶中(使用镊子)。或者, 用锋利的剃刀分开玫瑰花和根,把每个部分 植物在一个单独的小瓶。把小瓶放在方便 计数架,一旦你收获植物(为了避免 错配)。

  4. 将植物在烘箱中在50℃下干燥过夜

  5. 放射性测量
    1. 在化学罩下,加入2毫升闪烁鸡尾酒和紧 顶部在每个计数瓶。然后将瓶子放在适合的架子上 计数器
    2. 使用β计数器,在每个样品(Cs)和中计数 33


      在空白中(Cb是空白重复的平均值),也在 孵育培养基(C <10),10μl温育培养基, 计数小瓶和2ml闪烁混合物)。在里面 闪烁鸡尾酒,β辐射在光子中转化 由β计数器检测。度量单位为cpm(每分钟计数)。
    3. 数据分析:
      每小时每根厘米吸收的PO 4+的量(V,以nmol/h/cm计)如下计算:
      V =(Cs-Cb)*(S * 10 )* B /(T×L sub×C 10) /> Cs:样品中的放射性(cpm)
      Cb:空白中放射性的平均值(cpm)
      S:培养基中的P 1浓度(S =50μMPi) S * 10 -3 = 0.05:孵育培养基中的Pi含量(nmol /μl)(S =50μM)。 C 10:在10μl孵育溶液中的放射性(cpm)。 C 10 /(S * 10 -2 )是培养基中 P的比活性
      B = 10:在培育植物之前测量放射性的孵育溶液的体积(10μl是方便的)。
      T = 2:孵育2小时
      L :根长(cm)
      此外,使用与上面相同的方程,但省略L root ,你 可以计算每株植物的Pi吸收(Cs对应于33P中的 整个植物),每个玫瑰花结组织或根组织(Cs对应于33P 在叶子或根系统中)。
      这也是可能的 ?以计算在中测量的 33p(叶中的Cs,以cpm计)的比率 叶片相比于由全植株吸收的总的[33] ?根中的Cs(cpm)]。

代表数据

Pi 对于在低或高Pi上生长的植物,每小时和每根cm的摄取 (表格1)。呈现两种基因型(WT和突变体)以显示 植物之间Pi吸收的变化范围和分布 的植物中
表1.在低或高Pi吸收能力下生长的WT和突变体小植物中的Pi吸收和分布表示为每小时每根cm的nmol Pi(平均值±SD)。 Pi分布是由整个植物中的 33 P与 33 P的比率计算的。

食谱

  1. MS/10中等
    10倍稀释的Murashige和Skoog培养基,含有:
    0.15mM MgSO 4 2.1mM NH 4 NO 3
    1.9 mM KNO <3>
    0.5(高Pi)或0.005(低Pi)mM NaH 2 PO 4
    0.34mM CaCl 2·h/v 0.5μMKI
    10μMFeCl 2
    10μMH 3 BO 3
    10μMMnSO 4
    3μMZnSO 4
    0.1μMCuSO 4
    0.1μMCoCl 2
    1μMNa 2 MoO 4·
    5.9μM硫胺素 4.9μM吡哆素 8.1μM烟酸
    55μM肌醇
    3.4 mM MES
    0.5%蔗糖和0.8%琼脂,pH5.5
  2. 库存解决方案
    0.1mM CaCl 2在5mM MES中,在pH 5.5调节 对于1L(水):0.976g MES + 0.0147g CaCl 2,用10N NaOH调节pH至5.5 /
  3. 1 M KH 2 4
    对于1L(水):136g KH 2 PO 4 4 /
  4. 培养基(4ml /植物是必需的)
    50μMKH 2 PO 4:储备溶液中:50μl1M KH 2 PO 4在1L储备液中解决方案
    添加5,550Bq P/ml(0.15μCi P/ml)
  5. 解吸培养基(3ml /植物是必需的)
    1mM KH 2 PO 4在储备溶液中:1ml 1M KH 2 PO 4在1L储备液中解决方案

致谢

该方案改编自先前公开的研究:Narang等人(2000)和Misson等人(2004),并且由Ayadi等人。(2015)。这项工作得到了委员会原子能和替代能源支持。

参考文献

  1. Ayadi,A.,David,P.,Arrighi,J.F.,Chiarenza,S.,Thibaud,M.C.,Nussaume,L.and Marin,E。 减少拟南芥 PHOSPHATE TRANSPORTER1转运蛋白的遗传冗余度,以研究磷酸盐吸收和 167(4):1511-1526。
  2. Misson,J.,Thibaud,M.C.,Bechtold,N.,Raghothama,K.and Nussaume,L。(2004)。 拟南芥的转录调控和功能特性 Pht1; 4,高亲和力转运蛋白对磷酸盐剥夺植物中的磷酸盐摄取有很大的作用。植物分子生物学55(5):727-741。
  3. Narang,R.A.,Bruene,A。和Altmann,T。(2000)。 分析不同的拟南芥种质中的磷酸盐捕获效率。植物生理学 124(4):1786-1799。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Thibaud, M. and Marin, E. (2016). Measurement of 33P-PO4 Absorption Capacity and Root-to-leaf Transfer in Arabidopsis. Bio-protocol 6(5): e1741. DOI: 10.21769/BioProtoc.1741.
  2. Ayadi, A., David, P., Arrighi, J. F., Chiarenza, S., Thibaud, M. C., Nussaume, L. and Marin, E. (2015). Reducing the genetic redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 transporters to study phosphate uptake and signaling. Plant Physiol 167(4): 1511-1526.
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