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Measurement of 33P-PO4 Absorption Kinetic Constants in Arabidopsis
拟南芥吸收33P-PO4的动力学常数测定   

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Abstract

Based on the Michaelis-Menten kinetics model (Hofstee, 1952), this method allows calculation of the kinetic parameters (Vmax, Km) of phosphate uptake by Arabidopsis roots. This method is based on the quantification of phosphate uptake by Arabidopsis roots as described in Thibaud and Marin (2016) except that a range of phosphate concentration is applied in the incubation medium.
Plants are grown in high or low Pi giving access to kinetic parameters corresponding to low and high affinity respectively. In high Pi, the high-affinity transporters are not induced giving access to the low-affinity transport only. When plants are grown in low Pi, high affinity transporters are active, and the corresponding kinetic parameters can be measured. The calculation of Km and Vmax values is based on the Michaelis-Menten kinetics model.

Keywords: Phosphate absorption(磷的吸收), Kinetic constants(动力学常数), 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. 33P-PO4 5 mCi/ml (185 MBq/ml, 1.48-5.84 TBq/mg, >99% isotopically pure, less than 0.5 μM Pi) (PerkinElmer)
  8. Scintillation cocktail (PerkinElmer, Ultima GoldTM)
  9. MgSO4
  10. NH4NO3
  11. KNO3
  12. NaH2PO4
  13. Kl
  14. FeCl2
  15. MnSO4
  16. ZnSO4
  17. CuSO4
  18. CoCl2
  19. Na2MoO4
  20. Thiamine
  21. Pyridoxine
  22. Nicotinic acid
  23. Inositol
  24. Sucrose
  25. Agar
  26. MS/10 medium (see Recipes)
  27. Stock solution (see Recipes)
  28. 1 M KH2PO4 (Sigma-Aldrich) (see Recipes)
  29. Incubation medium (see Recipes)
  30. Desorption medium (see Recipes)

Equipment

  1. Liquid scintillation counter (PerkinElmer, Packard Instrument Company, model: TRI-CARB )
  2. Ice-containing large boxes for the desorption step (all 12-well plates will be placed horizontally on ice for 2 h)
  3. Tweezers for handling the plantlets
  4. Micropipets
  5. Shield for protection against radiations (plexiglass)
  6. 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)
  2. PRISM 6.0 software (GraphPad)

Procedure

  1. Preparation of plant materials
    9 to 11 day-old plantlets grown in high and low Pi 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 or 10 seeds are sown per plate depending on the growth medium (respectively with high or low Pi, see below). 9 or 8 plates will be necessary per plant type (respectively in high and low Pi).
    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. 12 plants per treatment (Pi concentration in the incubation medium) will be necessary plus 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 and 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 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 in Thibaud and Marin (2016).
    3. Prepare ten 12-well plates, one for each point of Pi concentration: 2, 5, 10, 20, 50, 100 μM for plants grown in low Pi and 200, 500, 1,000 and 2,000 μM for plants grown in high Pi. Number the wells: 1 to 12 (for 12 replicates) for each Pi concentration.
    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

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


    1. Prepare new 12-well plates, with 3 ml of cold desorption medium per well. Place the plates on ice.
    2. Rinse the 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 into the desorption medium for 2 h on ice.
    4. Transfer each plant into a counting vial (use tweezers). 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 in each counting vial and tight a top on it. 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=2 to 2,000 μM).
      S*10-3: Pi content (nmol/μl) in the incubation medium (S=2 to 2,000 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)
    4. Kinetics parameters are calculated by drawing Eadie-Hofstee plots where V (mean value of Pi uptake for each Pi concentration in the medium) is plotted against V/S (mean value, S=Pi concentration in the incubation medium). This representation reveals two uptake systems (high or low affinity, Figure 3). Kinetics parameters (Vmax and Km) are calculated by linear regression for each uptake system: Km is the slope of the equation and Vmax is the extrapolated value for V/S=0 (Table 1).
      Mean and standard deviation (SD) can be calculated from several experiments (not shown here).
    5. Kinetics parameters can also be calculated by nonlinear regression (based on Michaelis-Menten kinetics model on V values vs S) using PRISM 6.0 software (GraphPad) for high and low affinity systems separately (Table 1). With this procedure, mean and standard deviation (SD) of Vmax and Km can be calculated.

Representative data


Figure 3. Eadie-Hofstee plot showing the mean values for each Pi concentration uptake rates, the linear regression and the equations giving the Km and Vmax values for both low (in blue) and high (in black) Pi transport activity

Table 1. Vmax and Km values obtained with Eadie-Hofstee plot (from Figure 1) or with Prism software

Recipes

  1. MS/10 medium
    10x 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: 0.976 g MES + 0.0147 g CaCl2, Adjust pH to 5.5 with 10 N NaOH
  3. 1 M KH2PO4
    For 1 L: 136 g in water
  4. Incubation medium (4 ml /plant are necessary)
    5,550 Bq 33P/ml (0.15 µCi 33P/ml) in stock solution
    Then prepare aliquots with appropriate volume (see table below) of 1 M or 100 mM KH2PO4 for 2 to 2,000 µM final concentration in 40 ml stock solution supplied in 33P as follows:
    Final Pi concentration (µM)
     2
    5
    10
    20
    50
    100
    200
    500
    1,000
    2,000
    1 M KH2PO4 (µl)






    10
    20
    40
    80
    100 mM KH2PO4 (µl)
    0.8
    2
    4
    8
    20
    40





  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) modified by Misson et al. (2004) and Aung et al. (2006) based on the study of Hosftee (1952). We acknowledge all these authors for their previous work. The present protocol was published by Ayadi et al. (2015). This work was supported by the Commissariat à l’Energie Atomique et aux Energies Alternatives.

References

  1. Aung, K., Lin, S. I., Wu, C. C., Huang, Y. T., Su, C. L. and Chiou, T. J. (2006). pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene. Plant Physiol 141(3): 1000-1011.
  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.
  3. Hofstee, B. H. (1952). On the evaluation of the constants Vm and KM in enzyme reactions. Science 116(3013): 329-331.
  4. 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.
  5. Narang, R. A., Bruene, A. and Altmann, T. (2000). Analysis of phosphate acquisition efficiency in different Arabidopsis accessions. Plant Physiol 124(4): 1786-1799.
  6. Thibaud, M. and Marin, E. (2016). Measurement of 33P-PO4 absorption capacity and root-to-leaf transfer in Arabidopsis. Bio-protocol 6(5): e1741.

简介

基于Michaelis-Menten动力学模型(Hofstee,1952),该方法允许计算磷酸盐吸收的动力学参数(V max,K m) 拟南芥根。 该方法基于如Thibaud和Marin(2016)所述的拟南芥根的磷酸盐摄取的定量,除了在培养基中应用一定范围的磷酸盐浓度。
植物在高或低Pi中生长,提供分别对应于低亲和力和高亲和力的动力学参数。 在高Pi中,高亲和力转运蛋白不被诱导仅获得低亲和力转运。 当植物在低Pi中生长时,高亲和力转运蛋白是活性的,并且可以测量相应的动力学参数。 K m和V max值的计算基于Michaelis-Menten动力学模型。

关键字:磷的吸收, 动力学常数, 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:如果你想在根和叶中量化33P,每个植物需要一个小瓶,或者每个植物需要两个小瓶。在开始实验前,小瓶应从1到N编号。它们也应该按照适合于β计数器的适当机架(PerkinElmer)放置。
  3. 提示
  4. 年轻体外苗子
  5. MES水合物(Sigma-Aldrich,目录号:M8250)
  6. CaCl 2(Sigma-Aldrich)
  7. (185MBq/ml,1.48-5.84TBq/mg,> 99%同位素纯,小于0.5μMPi)(P <0.05),(P <0.05) PerkinElmer)
  8. 闪烁混合物(PerkinElmer,Ultima Gold TM
  9. MgSO 4 4 /
  10. NH 4 3
  11. KNO 3
  12. NaH 2 PO 4 sub
  13. Kl
  14. FeCl <2>
  15. MnSO 4
  16. ZnSO 4
  17. CuSO 4
  18. CoCl <2>
  19. Na MoO 4
  20. 硫胺素
  21. 吡哆醇
  22. 烟酸
  23. 肌醇
  24. 蔗糖
  25. Agar
  26. MS/10介质(见配方)
  27. 库存解决方案(参见配方)
  28. 1 M KH 2 PO 4(Sigma-Aldrich)(参见配方)
  29. 培养基(见配方)
  30. 解吸介质(参见配方)

设备

  1. 液体闪烁计数器(PerkinElmer,Packard Instrument Company,型号:TRI-CARB)
  2. 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)
  3. 用于处理苗的镊子
  4. 微信
  5. 防护辐射(有机玻璃)的盾牌
  6. 扫描仪或相机(爱普生美国,型号:Perfection V850Pro或佳能,型号:Powershot SX130),但其他制造商的其他设备可能适合

软件

  1. ImageJ版本1.46r与NeuronJ插件( http://imagej.nih.gov/ij
  2. PRISM 6.0软件(GraphPad)

程序

  1. 植物材料的制备
    生长在高和低Pi的9到11天的小植株便于实验
    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或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)。每个植物类型需要9或8个平板(分别为 高和低Pi)。
    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12株植物(培养基中的Pi浓度),加上空白的10株植物。
    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,照片大约为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 主根和侧根长度(参见图1图例中的方案)

      图1.根长度测量 A.比例尺的设置:使用ImageJ, 在方格纸上绘制1厘米刻度(1,2),然后"设置刻度"(3)。这个 给出像素/cm(4)。 B.根长的测量:用NeuronJ 插件,加载图像文件(5),然后设置缩放(如A中定义)。加 跟踪(6)通过沿根(粉红色痕迹,7)绘制然后测量 ?跟踪(RUN,8)。根长度(cm)(9)的结果可以复制到 ?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Thibaud和Marin(2016)所述的方案。
    3. 准备10个12孔板,每个点的Pi浓度:1, 5,10,20,50,100μM,对于在低Pi和200,500,1,000和200,000中生长的植物 ?对于在高Pi中生长的植物而言为2,000μM。井数:1到12(对于12 ?重复)每个Pi浓度
    4. 加入4 ml温育 培养基/孔。用镊子每孔放置1株植物(图2), 根应该被浸没和叶子在井外。避免 在孵化期间幼小植物的过度脱水, 小心地在板上盖上盖子,避免叶子的浸泡 在溶液中(图2B)。在室温下孵育2小时(22 ?-24℃)在白光下(150-180μE m <-200S -1 )。丢弃植物 ?完全浸没在液体中,如果有的话

      图2.孵化 实验。 A.植物编号为1至12英寸的12孔板的照片 培养基。为了清楚起见,移除了盖板。 请注意,玫瑰花是在媒介和根完全 浸没。 B.照片的板和盖(侧视图)显示如何 将盖子放置在12孔板上。

    5. 为了 评价Pi对根的吸附(机械或化学吸附 可以发生在细胞上,但在这种情况下,Pi不进入根内部 细胞),空白样品如下处理:浸泡植物根 在孵育培养基中2秒(使用镊子)和直接 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. 在水中冲洗植物(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 并将根在冰上浸没在解吸介质中2小时
    4. 将每个植物转移到计数瓶(使用镊子)。它是 方便在收获后立即将小瓶放在计数架上 ?植物(以避免错配)。

  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,每个添加2毫升闪烁鸡尾酒 计数瓶和紧紧上面。然后将小瓶放在机架中 适合计数器。
    2. 使用β计数器,每个计数 33 P 样品(Cs)和空白(Cb是空白复制品的平均值);和 也在孵育培养基(C <10)中,10μl的温育培养基是 置于计数小瓶中,加入2ml闪烁混合物)。在 ?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(count per min)。
    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Pi浓度(S = 2至2,000μM) (S = 2?2,000μM)中的Pi含量(nmol /μl)。< S/10< 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)

    4. 动力学参数通过绘制Eadie-Hofstee图计算 其中V(Pi中每个Pi浓度的吸收的平均值 介质)相对于V/S(平均值,S = 培养基)。这个表示揭示了两个摄取系统(高 ?或低亲和力,图3)。动力学参数(V sub max和K sub) 通过每个摄取系统的线性回归计算:K m是斜率 ,V max 是V/S = 0(表1)的外推值。
      平均值和标准偏差(SD)可以从几个实验(这里未示出)计算。
    5. 动力学参数也可以通过非线性回归计算 (基于Michaelis-Menten动力学模型对V值 S)使用PRISM 6.0软件(GraphPad)分别用于高亲和力和低亲和力系统 (表格1)。利用该过程,可以计算V max和K m的平均值和标准偏差(SD)。

代表数据


图3.Eadie-Hofstee图,其显示了每种Pi浓度吸收速率的平均值,线性回归和给出低(蓝色)和高(黑色)Pi转运活性的Km和Vmax值的方程式。/strong>

表1.使用Eadie-Hofstee图(来自图1)或使用Prism软件获得的V sub max和K sub的值

食谱

  1. MS/10中等
    10x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
    0.5μMKI
    10μMFeCl 2
    10μMH sub 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: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:136g在水中
  4. 培养基(4ml /植物是必需的)
    5,550 Bq P/ml(0.15μCi<33 P P/ml) 然后,在40ml储备溶液中,在40ml储备溶液中,用适当体积(参见下表)制备1μM或100mM KH 2 PO 4 4至2μM至2,000μM终浓度的等分试样, sup> 33 P,如下所示:
    最终Pi浓度(μM)
      2
    5
    10
    20
    50
    100
    200
    500
    1,000
    2,000
    1 M KH sub 2 PO 4 sub(μl)






    10
    20
    40
    80
    100mM KH 2 PO 4(μl)
    0.8
    2
    4
    8
    20
    40





  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

该方案改变自先前公开的研究,Narang等人(2000)由Misson等人修改。 (2004)和Aung等人(2006)基于Hosftee(1952)的研究。我们承认所有这些作者以前的工作。本议定书由Ayadi等人(2015)出版。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. Aung,K.,Lin,S.I.,Wu,C.C.,Huang,Y.T.,Su,C.L.and Chiou,T.J。(2006)。 pho2,一种磷酸盐过度积聚物,是由microRNA399靶基因中的无义突变引起的。 Plant Physiol 141(3):1000-1011。
  2. 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信号。植物生理学167(4):1511-1526。
  3. Hofstee,B.H。(1952)。 关于酶反应中常数Vm和KM的评估科学 116(3013):329-331。
  4. 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。
  5. 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
  6. Thibaud,M.和Marin,E.(2016)。 33 P-PO <4>吸收能力和根的测量到拟南芥中的叶至叶转移。 生物协议 6(5):e1741。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
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. Marin, E. and Thibaud, M. (2016). Measurement of 33P-PO4 Absorption Kinetic Constants in Arabidopsis. Bio-protocol 6(5): e1742. DOI: 10.21769/BioProtoc.1742.
  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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