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Quantification of Sodium Accumulation in Arabidopsis thaliana Using Inductively Coupled Plasma Optical Emission Spectrometery (ICP-OES)
采用电感耦合等离子体光发射谱法(ICP-OES)定量测定拟南芥中的钠累积   

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Abstract

Salt stress is a major issue for plants growing in both natural and agricultural settings (Deinlein et al., 2014). For example, irrigation can lead to the build up of salts in the soil as the irrigation water evaporates, leading to salinization, inhibition of plant growth, reduced productivity and eventually to loss of agriculturally usable land. One key element in trying to understand how salt stress impacts plant growth and development, in defining plant salt sensing and response mechanisms and eventually in the breeding or engineering of plants resistant to this stress is monitoring their salt uptake and redistribution. Methods such as imaging Na-sensitive fluorescent probes (Kader and Lindberg, 2005) and use of Na-ion selective microelectrodes (Shabala et al., 2005) offer the potential to follow Na levels in the plant in a non-destructive manner but are technically demanding and not applicable to field, or even many laboratory, conditions. However, tissue sampling followed by inductively coupled plasma spectroscopy (ICP) represents a simple, quantitative assay to monitor total Na levels in plant samples. ICP analysis is also applicable to plants in any environment where samples can be harvested. The approach uses tissue digestion in acid solutions, followed by injection of the resulting sample into an inductively coupled plasma spectrometer and monitoring the characteristic emitted spectrum from Na. As Na is stable, no complex sample preservation is required. Care needs to be taken with possible Na contamination in standards and samples from the water used for sample preparation and from glassware but otherwise, the approach is simple and robust.

Materials and Reagents

  1. Plant tissues
  2. 60% (vol/vol) Perchloric acid (HClO4) (Sigma-Aldrich, catalog number: 244252 )
  3. Nitric acid (HNO3) (Sigma-Aldrich, catalog number: 225711 )
  4. 18 megaohms (MΩ)-cm deionized water (DI water)
  5. NaCl standards, see Recipes for two methods of preparing Na standards
  6. ICP-OES calibration standard (Agilent, catalog number: 6610030700 )
  7. Kimwipe strip, W x H: 30 x 5 mm (manually prepared by cutting a single layer of Kimwipe) (Kimtech Science Kimwipers, catalog number: 34120 )
  8. NaCl standards (see Recipes)
  9. Alternative method of preparing Na standards from 1 M NaCl standard (see Recipes)
  10. Composition of plant growth medium (see Recipes)

Equipment

  1. Perkin-Elmer Optima 2000DV inductively coupled plasma optical emission spectrometer (ICP-OES) or similar machine
  2. Fume hood
  3. Heat block for heating glass test tubes
  4. Disposable borosilicate glass tubes with plain ends (O.D. x L: 16 x 125 mm) (Thermo Fisher Scientific, catalog number: 14-961-30 )
  5. Sterile square Petri dish with grid (L x W x H: 100 mm x 100 x 15 mm, grid of thirty-six 13 x 13 mm squares) (Thermo Fisher Scientific, catalog number: 08-757-11A )

Procedure

This experiment is designed to follow uptake of Na by the root tip and then its transport to the aerial parts of the seedling (Choi et al., 2014).

  1. Grow Arabidopsis wild type Columbia_0 (WT Col_0) on the surface of plant growth medium [including 0.5% (w/v) Phytagel] in 100 x 100 mm square Petri dishes for 10 days under long day conditions (16 h light/8 h dark).
  2. Cover the root tip (~5 mm of the root apex) for 1 h with a single layer Kimwipe strip (W x H: 30 x 5 mm) wetted with 50 µl of DI water (Figure 1A).
  3. After this acclimation period, soak the Kimwipe strip for a further 10 min with either 50 µl of DI water (control) or 50 µl of 100 mM NaCl (10 min salt stress).
  4. During these control and salt treatments, the Petri dishes should be raised ~15 mm at one end by placing them on a second empty Petri dish. This second Petri dish should be placed under the end of the Petri dish furthest from the seedling roots and closest to the cotyledon/shoot end of the plants (Figure 1B). This protocol ensures that the water or NaCl applied to the Kimwipe strip cannot passively flow along the surface of the plants toward the shoots.


    Figure 1. Salt stress experiment performed in a square Petri dish. A. Top view of 100 x 100 mm Petri dish with ten, 10 d old Arabidopsis (seedlings). The root tips are covered with a single layer Kimwipe strip (W x H: 30 x 5 mm). B. Side view of Petri dish with raised shoot end to prevent passive liquid flow to shoot tissue. The petri dish is covered with a lid to keep humidity high inside of the Petri dish.

  5. Quickly rinse the tissues to be analyzed in DI water to remove any Na passively adhering to their surface.
  6. Harvest ~50 mg of fresh tissues and record the weight of each sample. For example, to obtain samples of ~50 mg from roots or shoots you will need at least 10 seedlings of 8-10 day old Arabidopsis plants per sample. You will also need to plan for at least three independent experiments with three technical replicates per experiment for robust statistical analyses.
  7. In a fume hood, digest each sample in a separate glass test tube (washed with 1% (vol/vol) nitric acid and dried prior to tissue digestion) in 0.6 ml of nitric acid at 120-150 °C for ~2 h or until all the plant tissues are completely dissolved. The 120 °C temperature is readily maintained using a heat block.
  8. Add a further 0.4 ml of 60% (vol/vol) HClO4 and incubate at 150-180 °C for an additional 2 h or until the total sample volume is reduced to ≤ 0.5 ml. There is no need to constantly shake or mix the sample.
  9. Cool to room temperature and add DI water up to 5 ml.
  10. The sodium concentration can now be determined using a Perkin-Elmer Optima 2000DV inductively coupled plasma optical emission spectrometer (ICP-OES) or similar equipment according to the manufacturer’s instructions. For example, parameter settings for a Perkin-Elmer Optima 2000DV are 0.60 L/min Nebulizer flow rate, 1 ml/min sample flow rate and 4 ml of total sample usage for 4 replicates (1 ml of each replicate).
  11. Raw results of Na level can be converted to parts per million (milligrams per liter) using a standard curve (Figure 2). To make this standard curve, Na standard concentration values in ppm (mg/L) were plotted as the x-axis values and corresponding corrected/normalized Na intensity values (Table 2) were then input as the y-axis values. These data can then be converted to milligrams per kilogram fresh weight using the following equation (1):

  12. Data from Na standards are shown in Tables 1-2 and Figure 2. Typical data from this experiment are shown in Tables 3-6 and Figure 3:
    1. Table 1, raw Na intensity values obtained from Na standards.
    2. Table 2, corrected/normalized Na intensity values of Na standards using the obtained raw Na standard intensity values in the Table 1. Method for correction/normalization is described in the Table 2 ‘Note’.
    3. Figure 2, standard curve of corrected/normalized Na intensity values.
    4. Table 3, raw data of Na intensity values of the control and 10 min salt treated Arabidopsis samples.
    5. Table 4, corrected/normalized Na intensity values obtained from Table 3.
    6. Table 5, Na levels in ppm (mg/L) of the control and 10 min salt treated Arabidopsis seedlings. Conversion of Na intensity values to ppm (mg/L) was done using equation 2, which was obtained based on linear regression analysis of the corrected Na standards (Figure 2).

    7. Table 6, final conversion of Na levels in Table 5 to ‘mg/kg fresh weight (F.W.)’ using equation 1 (above step 11).
    8. Figure 3, comparison of Na level in mg/kg F.W. in the roots of 10 old Arabidopsis seedlings in response to 100 mM salt treatment.

Representative data

  1. Table 1. Table of raw intensity values from Na standards detected by ICP-OES
    Na concentration in standard (µM)
    Intensity value
    Replicate #1
    Replicate #2
    Replicate #3
    Replicate #4
    Average
    0 (DI water)
    -13101
    -12852
    -13607
    -13906
    -13367
    1
    62192
    62167
    62641
    62635
    62409
    10
    207932
    206271
    203571
    207676
    206363
    100
    8783775
    8703642
    8666576
    8517874
    8667967
    200
    20306803
    20237705
    20096115
    20508961
    20287396
    500
    63460855
    63572884
    64524580
    64081778
    63910024

    Table 2. Table of corrected* intensity values from raw Na standard values
    Na concentration in standard (µM)
    Corrected intensity value
    Replicate #1
    Replicate #2
    Replicate #3
    Replicate #4
    Average
    0 (DI water)
    265
    515
    -241
    -539
    0
    1
    75559
    75533
    76007
    76001
    303101
    10
    221299
    219637
    216938
    221043
    878916
    100
    8797141
    8717009
    8679942
    8531241
    34725332
    200
    20320170
    20251071
    20109481
    20522327
    81203049
    500
    63474222
    63586251
    64537946
    64095145
    255693563
    Note: *Na intensity is corrected/normalized by subtracting the average of the raw Na intensity values in the DI water from the raw Na intensity values in each Na standard. For example, in the above dataset, the average Na signal intensity values in DI water = ((-13101) + (-12852) + (-13607) + (-13906))/4 = -13367. Corrected/Normalized intensity value of replicate #1 of DI water is then -13101 - (-13367) = 265.

    The equation obtained from the representative Na standard curve in Figure 1 is Y = 5.545e+006X - 1.706e+006 where Y is the corrected Na signal intensity and X is the Na level in mg/L. Therefore, Na level in mg/L from an unknown sample can be calculated using equation 2.


    Figure 2. Na standard curve of corrected Na signal intensity. Na standard graph was generated using GraphPad Prism (Ver. 6) software (www.graphpad.com). Solid line represents corrected Na intensity (y axis) to corresponding Na concentration in ppm (mg/L, x axis). Dashed line indicates linear regression analysis of corrected/normalized Na standard (R2, 0.9912; slope, 5.545e+006 ± 111579; Y-intercept when X = 0, -1.706e+006 ± 573942; P value, < 0.0001).

    Table 3. Raw data of Na signal intensity values from control and 10 min salt stressed Arabidopsis seedlings

    Signal intensity
    Replicate #1
    Replicate #2
    Replicate #3
    Replicate #4
    Average
    Control root #1
    14268939.3
    14515870.1
    14259302.6
    14529114.1
    14393306.5
    Control root #2
    18390245.8
    18113970.6
    18376953.2
    18586787.6
    18366989.3
    Control root #3
    15961897.1
    15946196.2
    16018475.1
    16181451.1
    16027004.9
    10 min salt stress root #1
    16524276.7
    16462936.7
    16385847.1
    16373113.5
    16436543.5
    10 min salt stress root #2
    16710847.8
    16254854.6
    16242386.2
    16349709.2
    16389449.5
    10 min salt stress root #3
    16676297.9
    16596706.1
    17026112.0
    17105345.0
    16851115.3
    Note: Na detection using ICP-OES was repeated 4 times (4 technical replicates) from three independent biological replicates (roots #1-#3) for each treatment (for a total of 12 replicates).

    Table 4. Corrected/Normalized Na signal intensity values of raw data from Table 3

    Corrected/Normalizes signal intensity
    Replicate #1
    Replicate #2
    Replicate #3
    Replicate #4
    Average
    Control root #1
    14282305.8
    14529236.6
    14272669.1
    14542480.6
    14406673.0
    Control root #2
    18403612.3
    18127337.1
    18390319.7
    18600154.1
    18380355.8
    Control root #3
    15975263.6
    15959562.7
    16031841.6
    16194817.6
    16040371.4
    10 min salt stress root #1
    16537643.2
    16476303.2
    16399213.6
    16386480.0
    16449910.0
    10 min salt stress root #2
    16724214.3
    16268221.1
    16255752.7
    16363075.7
    16402816.0
    10 min salt stress root #3
    16689664.4
    16610072.6
    17039478.5
    17118711.5
    16864481.8

    Table 5. Conversion of Na signal intensity values to concentration in ppm (mg/L)

    Na level (mg/L)
    replicate #1
    replicate #2
    replicate #3
    replicate #4
    Average
    Standard deviation (SD)
    Control root #1
    2.883
    2.928
    2.882
    2.930
    2.91
    0.027
    Control root #2
    3.627
    3.577
    3.624
    3.662
    3.62
    0.035
    Control root #3
    3.189
    3.186
    3.199
    3.228
    3.20
    0.019
    10 min salt stress root #1
    3.290
    3.279
    3.265
    3.263
    3.27
    0.013
    10 min salt stress root #2
    3.324
    3.242
    3.239
    3.259
    3.27
    0.040
    10 min salt stress root #3
    3.318
    3.303
    3.381
    3.395
    3.35
    0.045
    Note: Na intensity from the samples can be converted into ppm (mg/L) using the equation 2.

    Table 6. Conversion of Na level to ‘mg/kg fresh weight (F.W.)

    Na level (mg/kg F.W.)
    replicate #1
    replicate #2
    replicate #3
    replicate #4
    Average
    Standard deviation
    (SD)
    Control root #1  
     2151.8   
    2185.0   
    2150.5   
    2186.8   
    2168.51   
    20.096
    Control root #2
    2706.4
    2669.3
    2704.6
    2732.9
    2703.30
    26.116
    Control root #3
    2379.6
    2377.5
    2387.2
    2409.2
    2388.38
    14.473
    10 min salt stress root #1
    2611.2
    2602.4
    2591.4
    2589.6
    2598.64
    10.116
    10 min salt stress root #2
    2518.0
    2455.7
    2454.0
    2468.7
    2474.08
    29.997
    10 min salt stress root #3
    2047.9
    2039.0
    2086.8
    2095.6
    2067.31
    28.055
    Note: Na level in ppm (mg/L) in Table 5 was converted to Na level in ‘mg/L fresh weight’ using the equation 1.


    Figure 3. Comparison of Na level (mg/kg F.W.) in the roots of 10d old Arabidopsis in response to salt stress. Root tips of 10 d old Arabidopsis seedlings were subjected to salt stress with 100 mM NaCl for 10 min as described above. Error bar, standard deviation of n = 12 (P < 0.001). ***Student's t-test p value < 0.001 compared to Control.

    Notes

    1. Some sources of water contain high levels of Na that will provide a background to each sample. It is important to run a calibration on the ICP machine being used with an ICP-OES calibration standard according to the manufacturer's instructions. One commercial calibration mix for ICP-OES from Agilent Technologies, contains 500 mg/L Ca, Fe, K, Mg and Na in 5% (v/v) HNO3. Once the machine is calibrated, measure a Na calibration curve (see Recipes), paying special attention to the 0 Na point to establish if significant Na contamination is occurring. Using DI water is essential but contamination may also occur if the equipment has been previously used for samples with high Na content. Thorough flushing of the equipment with DI water is needed under these circumstances.
    2. The provided exemplary datasets in this protocol were obtained from 10 day old Arabidopsis wild type Columbia-0 ecotype seedlings grown under long day light cycles (16 h light/8 h dark) at 22 °C. Therefore, Na accumulation values might be variable depending on plant species, growth conditions, age, and genotypes.

    Recipes

    1. NaCl standards
      Na (μM)
      NaCl (g/L)
      0
      0
      1
      0.000023
      10
      0.00023
      100
      0.0023
      200
      0.0046
      500
      0.0115
      Note: Dissolve NaCl in DI water as indicated.
    2. Alternative method of preparing Na standards from 1 M NaCl standard
      1. Prepare 1 ml of 1 M Na standard solution by dissolving 0.023 g of NaCl into 1 ml DI water and vortex until NaCl is completely dissolved.
      2. Dilute 1 M NaCl standard as below.
        NaCl standard
        Volume of NaCl standard
        Volume of DI water
        Dilution factor
        Total Volume (ml)
        10 mM
        10 µl of 1 M NaCl
        990 µl
        1:100
        1 ml
        1 mM
        1 ml of 10 mM NaCl
        9 ml
        1:10
        10 ml
        500 µM
        5 ml of 1 mM NaCl
        5 ml
        1:1
        10 ml
        200 µM
        2 ml of 1 mM NaCl
        8 ml
        1:5
        10 ml
        100 µM
        1 ml of 1 mM NaCl
        9 ml
        1:10
        10 ml
        10 µM
        0.1 ml of 1mM NaCl
        9.9 ml
        1:100
        10 ml
        1 µM
        0.1 ml of 100 µM NaCl
        9.9ml
        1:100
        10 ml
        Note: Make sure to make a minimum 5 ml of NaCl standard solutions as one measurement uses 1 ml of each standard solution but replicates for each point are needed.
    3. Composition of plant growth medium
      The plant growth medium is half strength Epstein medium consisting of 3 mM KNO3, 2 mM Ca(NO3)2.4H2O, 0.5 mM MgSO4.7H2O, 1 mM (NH4)H2PO4, 0.56 mM myo-inositol, 2.3 mM 2-(N-morpholino)ethanesulfonic acid (MES), 10 mM sucrose, micro-nutrients, and 0.5% (w/v) Phytagel at pH 5.7.
      The micro-nutrients are 25 μM KCl, 17.5 μM H3BO3, 1 µM MnSO4.H2O, 1 µM ZnSO4.7H2O, 0.25 µM CuSO4.5H2O, 0.25 µM (NH4)6MoO24.4H2O, and 25 µM (ethylene-dinitrilo)tetraacetic acid (Fe-Na EDTA).

    Acknowledgments

    The authors gratefully acknowledge funding from National Aeronautics and Space Administration (NNX13AM50G) and the National Science Foundation (NSF IOS-11213800, MCB-1329723) that supports this work. This protocol was adapted from Lahner et al. (2003) with slightly modification for small sample size of the use of fresh tissues.

    References

    1. Choi, W. G., Toyota, M., Kim, S. H., Hilleary, R. and Gilroy, S. (2014). Salt stress-induced Ca2+ waves are associated with rapid, long-distance root-to-shoot signaling in plants. Proc Natl Acad Sci U S A 111(17): 6497-6502.
    2. Deinlein, U., Stephan, A. B., Horie, T., Luo, W., Xu, G. and Schroeder, J. I. (2014). Plant salt-tolerance mechanisms. Trends Plant Sci 19(6): 371-379.
    3. Kader, M. A. and Lindberg, S. (2005). Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI. J Exp Bot 56(422): 3149-3158.
    4. Lahner, B., Gong, J., Mahmoudian, M., Smith, E. L., Abid, K. B., Rogers, E. E., Guerinot, M. L., Harper, J. F., Ward, J. M., McIntyre, L., Schroeder, J. I. and Salt, D. E. (2003). Genomic scale profiling of nutrient and trace elements in Arabidopsis thaliana. Nat Biotechnol 21(10): 1215-1221.
    5. Shabala, L., Cuin, T. A., Newman, I. A. and Shabala, S. (2005). Salinity-induced ion flux patterns from the excised roots of Arabidopsis sos mutants. Planta 222(6): 1041-1050.

简介

盐胁迫是对于在自然和农业环境中生长的植物的主要问题(Deinlein等人,2014)。例如,当灌溉水蒸发时,灌溉可导致土壤中盐的积累,导致盐化,抑制植物生长,降低生产力并最终导致农业上可用的土地损失。试图了解盐胁迫如何影响植物生长和发育,确定植物盐感测和响应机制以及最终在抗这种胁迫的植物育种或工程中的一个关键因素是监测它们的盐吸收和再分布。诸如成像Na敏感性荧光探针(Kader和Lindberg,2005)和使用Na离子选择性微电极(Shabala等人,2005)的方法提供了在植物中遵循Na水平的潜力非破坏性的方式,但是在技术上要求和不适用于现场,或甚至许多实验室条件。然而,组织取样,随后电感耦合等离子体光谱(ICP)代表了监测植物样品中的总Na水平的简单,定量测定。 ICP分析也适用于可以收获样品的任何环境中的植物。该方法使用酸溶液中的组织消化,随后将所得样品注入电感耦合等离子体光谱仪并监测来自Na的特征发射光谱。由于Na是稳定的,不需要复杂的样品保存。需要注意来自用于样品制备的水和来自玻璃器皿的标准品和样品中可能的Na污染,否则,该方法简单且稳定。

材料和试剂

  1. 植物组织
  2. 60%(vol/vol)高氯酸(HClO 4)(Sigma-Aldrich,目录号:244252)
  3. 硝酸(HNO 3)(Sigma-Aldrich,目录号:225711)
  4. 18兆欧(MΩ)-cm去离子水(去离子水)
  5. NaCl标准,参见两种制备Na标准的方法的配方
  6. ICP-OES校准标准品(Agilent,目录号:6610030700)
  7. Kimwipe条,W×H:30×5mm(通过切割单层Kimwipe手工制备)(Kimtech Science Kimwipers,目录号:34120)
  8. NaCl标准(见配方)
  9. 从1M NaCl标准品制备Na标准品的替代方法(参见配方)
  10. 植物生长培养基的组成(见配方)

设备

  1. Perkin-Elmer Optima 2000DV电感耦合等离子体发射光谱仪(ICP-OES)或类似机器
  2. 通风橱
  3. 加热玻璃试管的加热块
  4. 具有平端的一次性硼硅酸盐玻璃管(O.D.xL:16×125mm)(Thermo Fisher Scientific,目录号:14-961-30)
  5. 将具有网格(L×W×H:100mm×100×15mm,三十六个13×13mm正方形的网格)(Thermo Fisher Scientific,目录号:08-757-11A)的无菌方形陪替氏培养皿

程序

该实验设计为遵循根尖吸收Na,然后将其转运到幼苗的地上部分(Choi等人,2014)。

  1. 在表面上生长拟南芥野生型Columbia_0(WT Col_0) 植物生长培养基[包括0.5%(w/v)Phytagel]在100×100mm 在长日照条件下(16小时光照/8小时)平皿培养10天   黑暗)。
  2. 覆盖根尖(〜5毫米的根尖)1小时   用50μlDI润湿的单层Kimwipe条(W×H:30×5mm) 水(图1A)。
  3. 在这个适应期后,浸泡 Kimwipe条用50μl去离子水再洗10分钟 (对照)或50μl100mM NaCl(10分钟盐胁迫)。
  4. 中 这些控制和盐处理,培养皿应提高〜15  mm,将它们放在第二个空培养皿中。这第二个  培养皿应该放在培养皿的末端最远 从幼苗根中最接近子叶/芽端 植物(图1B)。此协议确保水或NaCl应用  到Kimwipe条不能被动地沿着表面流动 植物朝向芽。


    图1.盐胁迫实验 在方形培养皿中进行。 A. 100×100mm培养皿的顶视图  与10,10 d old拟南芥(seedyl)。根尖覆盖 与单层Kimwipe条(W×H:30×5mm)。 B.侧视图 培养皿与凸起的射击结束,以防止被动液体流动射击  组织。培养皿覆盖有盖子以保持湿度高 培养皿内。

  5. 在去离子水中快速冲洗待分析的组织,以去除被动粘附在其表面的任何Na
  6. 收获〜50毫克的新鲜组织,并记录每个的重量 样品。 例如,从根或枝条获得〜50mg的样品 您将需要至少10株8-10天的拟南芥植物 每个样品。 你还需要计划至少三个独立 实验三个技术重复每个实验为鲁棒 统计分析
  7. 在通风橱中,消化每个样品 单独的玻璃试管(用1%(vol/vol)硝酸洗涤并干燥   在组织消化之前)在0.6ml硝酸中在120-150℃下培养〜2小时或直到所有植物组织完全溶解。 120℃   使用加热块容易维持温度
  8. 添加 进一步0.4ml的60%(vol/vol)HClO 4,并在150-180℃下孵育 另外2小时或直到总样品体积减少至≤0.5ml。 无需不断摇晃或混合样品。
  9. 冷却至室温,加入去离子水至5ml。
  10. 现在可以使用Perkin-Elmer测定钠浓度   Optima 2000DV电感耦合等离子体发射光谱仪 (ICP-OES)或类似设备 说明。 例如,Perkin-Elmer Optima的参数设置 2000DV为0.60L/min雾化器流速,1ml/min样品流速和   4个总样品用量为4次重复(每次重复1ml)
  11. Na水平的原始结果可以转换为百万分之几 (毫克/升)使用标准曲线(图2)。 做到这一点 标准曲线,Na标准浓度值(mg/L) 绘制为x轴值和对应的校正/归一化的Na 强度值(表2)作为y轴值。 这些 数据然后可以使用转化为每千克鲜重的毫克   (1):

  12. Na标准的数据是 如表1-2和图2所示。该实验的典型数据为 如表3-6和图3所示:
    1. 表1,从Na标准品获得的原始Na强度值。
    2. 表2,Na标准品的校正/归一化Na强度值 使用获得的表1中的原始Na标准强度值。 表2"注"中描述了校正/归一化的方法
    3. 图2,校正/归一化Na强度值的标准曲线
    4. 表3,对照和10分钟盐处理的拟南芥样品的Na强度值的原始数据。
    5. 表4,从表3获得的校正/归一化Na强度值
    6. 表5,对照和10分钟盐的Na含量(ppm)(mg/L) 处理的拟南芥幼苗。 Na强度值转换为ppm (mg/L)使用基于线性获得的等式2进行 校正Na标准物的回归分析(图2)。

    7. 表6,使用方程式1(上述步骤11)将表5中的Na水平的最终转化为"mg/kg鲜重(F.W.)"。
    8. 图3,Na含量以mg/kg F.W.在根中的比较10 响应于100mM盐处理的拟南芥幼苗。

代表数据

  1. 表1. ICP-OES 检测到的Na标准的原始强度值表。
    Na浓度(μM)
    强度值
    复制#1
    复制#2
    复制#3
    复制#4
    平均
    0(去离子水)
    -13101
    -12852
    -13607
    -13906
    -13367
    1
    62192
    62167
    62641
    62635
    62409
    10
    207932
    206271
    203571
    207676
    206363
    100
    8783775
    8703642
    8666576
    8517874
    8667967
    200
    20306803
    20237705
    20096115
    20508961
    20287396
    500
    63460855
    63572884
    64524580
    64081778
    63910024

    表2.原始Na标准值
    的校正*强度值表
    Na浓度(μM)
    校正强度值
    复制#1
    复制#2
    复制#3
    复制#4
    平均
    0(去离子水)
    265
    515
    -241
    -539
    0
    1
    75559
    75533
    76007
    76001
    303101
    10
    221299
    219637
    216938
    221043
    878916
    100
    8797141
    8717009
    8679942
    8531241
    34725332
    200
    20320170
    20251071
    20109481
    20522327
    81203049
    500
    63474222
    63586251
    64537946
    64095145
    255693563
    注意: * Na强度通过减去   来自原始Na强度的去离子水中的原始Na强度值 值。 例如,在上述数据集中, DI水中的平均Na信号强度值=((-13101)+(-12852)+ (-13607)+(-13906))/4 = -13367。 校正/归一化强度值   重复#1的去离子水然后是-13101 - (-13367)= 265。

    的 方程式从图1中的代表性Na标准曲线获得 是Y = 5.545×e + 006×-1.706e + 006 其中Y是校正的Na信号 强度,X是Na水平,mg/L。因此,Na水平以mg/L为单位 从未知样品可以使用公式2计算

    图2.校正的Na信号强度的Na标准曲线。 Na 标准图使用GraphPad Prism(Ver.6)软件产生 ( www.graphpad.com )。实线表示校正的Na强度(y 轴)至相应的Na浓度(ppm)(mg/L,x轴)。虚线 线表示校正/归一化Na的线性回归分析 标准(R sup 2,0.9912;斜率,5.545e + 006 ±111579;当X = 0时的Y截距, -1.706e <+ sup> +006 ±573942; P 值, 0.0001)。

    表3.来自对照和10分钟盐胁迫的拟南芥苗的Na信号强度值的原始数据

    信号强度
    复制#1
    复制#2
    复制#3
    复制#4
    平均
    控制根#1
    14268939.3
    14259302.6

    14393306.5
    控制根#2

    18113970.6
    18776953.2

    18366989.3
    控制根#3
    15961897.1
    15946196.2
    16018475.1
    16181451.1
    16027004.9
    10分钟盐胁迫根#1

    16462936.7

    16373113.5
    16436543.5
    10分钟盐胁迫根#2
    16710847.8
    16254854.6
    16242386.2
    16349709.2

    10分钟盐胁迫根#3
    16776297.9

    17026112.0
    17105345.0
    16851115.3
    注意:   使用ICP-OES的Na检测重复4次(4次技术 重复)从三个独立的生物复制品(根#1-#3) 每次治疗(共12次重复)。

    表4.来自表3的原始数据的校正/归一化Na信号强度值

    校正/规范信号强度
    复制#1
    复制#2
    复制#3
    复制#4
    平均
    控制根#1
    14282305.8
    14529236.6
    14272669.1
    14542480.6
    14406673.0
    控制根#2
    18403612.3
    18127337.1

    18600154.1

    控制根#3
    15975263.6
    15959562.7
    16031841.6
    16194817.6
    16040371.4
    10分钟盐胁迫根#1
    16537643.2
    16476303.2
    16799213.6
    16386480.0
    16449910.0
    10分钟盐胁迫根#2
    16724214.3
    16268221.1
    16255752.7
    16363075.7
    16402816.0
    10分钟盐胁迫根#3
    16689664.4
    16610072.6
    17039478.5
    174118711.5
    16864481.8

    表5. Na信号强度值与ppm(mg/L)浓度的转换
    0.019
    ...

    Na水平(mg/L)
    复制#1
    复制#2
    复制#3
    复制#4
    平均
    标准偏差(SD)
    控制根#1
    2.883
    2.928
    2.882
    2.930
    2.91
    0.027
    控制根#2
    3.627
    3.577
    3.624
    3.662
    3.62
    0.035
    控制根#3
    3.189
    3.186
    3.199
    3.228
    3.20
    0.019
    10分钟盐胁迫根#1
    3.290
    3.279
    3.265
    3.263
    10分钟盐胁迫根#1
    3.290
    3.279
    3.265
    3.263
    3.259
    3.27
    0.040
    10 min salt stress root #3
    3.318
    3.303
    3.381
    3.395
    3.35
    0.045
    Note: Na intensity from the samples can be converted into ppm (mg/L) using the equation 2.

    Table 6. Conversion of Na level to ‘mg/kg fresh weight (F.W.)

    Na level (mg/kg F.W.)
    replicate #1
    replicate #2
    复制#3
    复制#4
    平均
    标准偏差
    (SD)
    控制根#1  
      2151.8   
    2185.0   
    2150.5   
    2186.8   
    2168.51   
    20.096
    控制根#2
    2706.4
    2669.3
    2704.6
    2732.9
    2703.30
    26.116
    控制根#3
    2379.6
    2377.5
    2387.2
    2409.2
    2388.38
    14.473
    10分钟盐胁迫根#1
    2611.2
    2602.4
    2591.4
    2589.6
    2598.64
    10.116
    10分钟盐胁迫根#2
    2518.0
    2455.7
    2454.0
    2468.7
    2474.08
    29.997
    10分钟盐胁迫根#3
    2047.9
    2039.0
    2086.8
    2095.6 / 2067.31
    28.055
    注意:使用公式1将表5中的以ppm(mg/L)表示的Na水平换算为"mg/L鲜重"中的Na水平。


    图3.在响应盐胁迫时10d旧的拟南芥根中Na水平(mg/kg FW)的比较。将10天的拟南芥幼苗的根尖进行盐 应力与100mM NaCl 10分钟,如上所述。错误栏, n = 12的标准偏差(P <0.001)。 ***学生t检验p值 < 0.001与对照相比。

    笔记

    1. 一些水源含有高水平的Na,其将为每个样品提供背景。重要的是,根据制造商的说明,使用ICP-OES校准标准对ICP机器进行校准。来自Agilent Technologies的ICP-OES的一种商业校准混合物在5%(v/v)HNO 3中含有500mg/L Ca,Fe,K,Mg和Na。一旦机器校准,测量Na校准曲线(参见配方),特别注意0 Na点,以确定是否发生显着的Na污染。使用去离子水是必要的,但如果设备以前用于具有高Na含量的样品,也可能发生污染。在这种情况下,需要用去离子水彻底冲洗设备
    2. 本方案中提供的示例性数据集是从22天在长日光循环(16小时光照/8小时黑暗)下生长的10日龄拟南芥野生型Columbia-0生态型幼苗获得的。 因此,Na积累值可能根据植物物种,生长条件,年龄和基因型而变化。

    食谱

    1. NaCl标准
      Na(μM)
      NaCl(g/L)
      0
      0
      1
      0.000023
      10
      0.00023
      100
      0.0023
      200
      0.0046
      500
      0.0115
      注意:按照指示将NaCl溶解在去离子水中。
    2. 从1M NaCl标准品制备Na标准品的另一种方法
      1. 准备1毫升的1M Na标准溶液通过溶解0.023克 NaCl溶于1ml去离子水中并涡旋,直到NaCl完全溶解
      2. 稀释1 M NaCl标准溶液,如下所示
        NaCl标准
        NaCl标准体积
        去离子水的体积
        稀释系数
        总体积(ml)
        10 mM
        10μl的1M NaCl
        990微升
        1:100
        1 ml
        1 mM
        1ml 10mM NaCl 9 ml
        1:10
        10 ml
        500μM
        5ml 1mM NaCl 5 ml
        1:1
        10 ml
        200μM
        2ml 1mM NaCl
        8 ml
        1:5
        10 ml
        100μM
        1ml 1mM NaCl
        9 ml
        1:10
        10 ml
        10μM
        0.1ml 1mM NaCl 9.9毫升
        1:100
        10 ml
        1μM
        0.1ml100μMNaCl
        9.9毫升
        1:100
        10 ml
        注意:   确保最少5毫升NaCl标准溶液作为一个 测量使用1ml的每种标准溶液,但对每种标准溶液进行复制 点。
    3. 植物生长培养基的组成
      植物生长培养基是由3mM KNO 3,2mM Ca(NO 3)2 Sub 2+组成的半强度Epstein培养基。/NH 4 H 2 O,0.5mM MgSO 4 .7H 2 O 2,1mM(NH 微量营养物是25μMKCl,17.5μMH 3 BO 3 Sub,1μMMnSO 4 sub。 H 2μM,1μMZnSO 4 sub,7H 2 O 2,0.25μMCuSO 4 sub/(NH 4)6 MoO 24子午线,0.25μM(NH 4)6 MoO 24子午线, 4H 2 O和25μM(乙烯 - 二次氮基)四乙酸(Fe-Na EDTA)。

    致谢

    作者衷心感谢国家航空航天局(NNX13AM50G)和国家科学基金会(NSF IOS-11213800,MCB-1329723)的资助,支持这项工作。该方案改编自Lahner等人(2003),对于使用新鲜组织的小样本尺寸略微修改。

    参考文献

    1. Choi,W.G.,Toyota,M.,Kim,S.H.,Hilleary,R.and Gilroy,S。(2014)。 盐胁迫诱导的Ca 2 + 波与快速,长期 - 植物中的根至茎信号。美国国家科学院院刊111(17):6497-6502。
    2. Deinlein,U.,Stephan,A.B.,Horie,T.,Luo,W.,Xu,G.and Schroeder,J.I。(2014)。 植物耐盐机制 趋势植物科学 19 (6):371-379。
    3. Kader,M.A。和Lindberg,S。(2005)。 在盐的盐敏感和耐盐性栽培品种的原生质体中摄取钠,Oryza由荧光染料SBFI确定的 L. 56(422):3149-3158。
    4. Lahner,B。,Gong,J.,Mahmoudian,M.,Smith,EL,Abid,KB,Rogers,EE,Guerinot,ML,Harper,JF,Ward,JM,McIntyre,L.,Schroeder, DE(2003)。 拟南芥中营养和微量元素的基因组规模分析。 a> Nat Biotechnol 21(10):1215-1221
    5. Shabala,L.,Cuin,T.A.,Newman,I.A。和Shabala,S。(2005)。 拟南芥切根的盐诱导离子通量模式 > sos 突变体。 Planta 222(6):1041-1050。

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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Choi, W. and Gilroy, S. (2015). Quantification of Sodium Accumulation in Arabidopsis thaliana Using Inductively Coupled Plasma Optical Emission Spectrometery (ICP-OES). Bio-protocol 5(16): e1570. DOI: 10.21769/BioProtoc.1570.
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