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Nitrate Assay for Plant Tissues

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The Plant Cell
Feb 2016



Nitrogen is an essential macronutrient for plant growth and nitrate content in plants can reflect the nitrogen supply of soil. Here, we provide the salicylic acid method to evaluate the nitrate content in plant tissues. The method is reliable and stable, thus it can be a good choice for measurement of nitrate in plant tissues.

Keywords: Nitrate content (硝酸盐含量), Plant (植物), Salicylic acid-sulphuric acid (水杨酸 - 硫酸), NaOH (NaOH), Standard curve (标准曲线), Boil (消煮), OD410 (OD410)


Nitrogen is an important macronutrient required by plants for normal growth and development. Usually most plants absorb nitrogen mainly in the form of nitrate grown under aerobic conditions (Xu et al., 2016). To determine the nitrate accumulation in plants, we need to test the nitrate content in different tissues of plants. There are some methods for determination of nitrate, for example, potentiometric method (Carlson and Keeney, 1971), phenoldisulfonic acid method (Bremner, 1965), Cadium reduction (Huffman and Barbarick, 1981) and other methods. These methods have some disadvantages, such as lower sensitivity, interferences, technician exposure to carcinogenic chemicals (Cataldo et al., 1975; Vendrell and Zupancic, 1990)

Here, we provide the salicylic acid method that is free of interferences, reliable and stable. Nitrosalicylic acid is formed by the reaction of nitrate and salicylic acid under highly acidic conditions. The complex is yellow under basic (pH > 12) condition with maximal absorption at 410 nm. The absorbance is directly proportional to nitrate content. Therefore the nitrate content in tissues can be calculated based on their absorbances. This method is suitable for determination of nitrate concentration in plants.

Materials and Reagents

  1. 1.5 ml Eppendorf tubes
  2. 12-ml plastic culture tube (Greiner Bio One, catalog number: 184261 )
  3. Quartz cuvettes  
  4. Arabidopsis thaliana roots and/or shoots (7-day-old seedlings)
  5. Potassium nitrate (KNO3) (Sinopharm Chemical Reagent, catalog number: 10017218 )
  6. Deionized water
  7. MS medium
  8. Liquid nitrogen
  9. Salicylic acid (Sinopharm Chemical Reagent, catalog number: 30163517 )
  10. Sulphuric acid (98%) (Sinopharm Chemical Reagent, catalog number: 100216008 )
  11. Sodium hydroxide (NaOH) (Sinopharm Chemical Reagent, catalog number: 10019718 )
  12. 500 mg/L (0.0357 mol/L) KNO3 standard solution (see Recipes)
  13. 5% (w/v) salicylic acid-sulphuric acid (see Recipes)
  14. 8% (w/v) NaOH solution (see Recipes)


  1. 50 ml flask
  2. Frozen mixed ball grinding machine (RETCH, model: MM400 )
  3. Visible light spectrophotometer (PGENERAL, catalog number: T6 )
  4. Centrifuge (Eppendorf, model: 5424 )


  1. Excel


  1. Standard curve
    1. To make the standard curve, 1 ml, 2 ml, 3 ml, 4 ml, 6 ml, 8 ml, 10 ml, and 12 ml NO3- standard solution (500 mg/L) is transferred to eight 50 ml flasks respectively, and deionized water is added to each solution to bring the total volume to 50 ml. The concentration of the series of standard solution should be 10, 20, 30, 40, 60, 80, 100, and 120 mg/L, respectively. And the molarity of 10, 20, 30, 40, 60, 80, 100, and 120 mg/L KNO3 is 0.0007, 0.0014, 0.0021, 0.0029, 0.0043, 0.0057, 0.0071, 0.0086 mol/L, respectively.
    2. Transfer 0.1 ml of each standard solution into a 12-ml tube, respectively. Use 0.1 ml deionized water as a control.
    3. Add 0.4 ml salicylic acid-sulphuric acid into each tube and mix well, and then incubate all reactions at room temperature for 20 min.
    4. Add 9.5 ml of 8% (w/v) NaOH solution into each tube, cool down the tubes (heat is generated due to the reaction) to room temperature (about 20-30 min), and measure the OD410 value with the control for reference.
    5. Plot the standard curve with the nitrate concentration as the horizontal axis and the absorbance as the vertical axis. Then, the regression equation can be obtained based on the standard curve (Figure 1). The detailed methods are as follows:
      1. Open an Excel, enter the OD410 values in column A and the nitrate concentrations of the standard solutions in column B. Select all the cells containing values, and then insert a scatter plot.
      2. Select any data point in the plot, right click, select to add a trend line, choose the linear and display equation, then standard curve and the regression equation are obtained.

      Figure 1. Standard curve. The 10, 20, 30, 40, 60, 80, 100, and 120 mg/L standard solutions are used to establish a standard curve. Error bars represent SD of biological replicates (n = 4). According to the standard curve, the regression equation is C (µg/ml) = 140.86 x OD410 - 1.1831, where C stands for nitrate concentration.

  2. Nitrate assay in Arabidopsis
    1. The seedlings are grown on half MS medium for 7 days (as shown in Figure 2), and the seedlings, shoots, and roots are collected separately for nitrate content determination.

      Figure 2. Hydroponic cultivation system for Arabidopsis seedlings. A. Arabidopsis seeds are grown on a gauze net (250 microns mesh size) that has been sterilized by autoclaving. B. The gauze net is placed on a bracket. Make sure that the medium level in the beaker reaches to the gauze net.

    2. Freeze each weighed sample (≤ 0.1 g, for example, about 20-25 7-day-old wildtype seedlings grown on half MS) in a 1.5-ml tube by liquid nitrogen, and grind each sample into powder with the frequency of 30/sec for 1 min using a RETCH MM400.
    3. Add 1 ml deionized water into the tubes and boil at 100 °C for 20 min (at least).
    4. Centrifuge the samples at 15,871 x g for 10 min, and transfer 0.1 ml supernatant into a new 12-ml tube. Use 0.1 ml deionized water as a control.
    5. Add 0.4 ml salicylic acid-sulphuric acid into each tube, mix the sample well, and then incubate the reactions for 20 min at room temperature.
    6. Add 9.5 ml of 8% (w/v) NaOH solution into each tube and cool down the tubes to room temperature (about 20-30 min). Measure the OD410 value of each sample with the control for reference.
    7. According to the OD410 value obtained in the above step, calculate the nitrate concentration (C) with the regression equation, C (µg/ml) = 140.86 x OD410 - 1.1831 obtained in the Procedure A (Figure 1).
    8. Calculate the nitrate content using the following equation:
      Y = CV/W
      Y: nitrate content (µg/g),
      C: nitrate concentration calculated with OD410 into regression equation as step B7 (µg/ml),
      V: the total volume of extracted sample (ml),
      W: weight of sample (g).

Data analysis

Table 1. The nitrate content of the roots of WT. Seedlings were grown on half MS medium for 7 days and the roots were collected for nitrate determination.

Note: The other results of nitrate content in plant tissues were published in the paper of ‘The Arabidopsis NRG2 protein mediates nitrate signaling and interacts with and regulates key nitrate regulators’ (http://www.plantcell.org/content/28/2/485.long).


  1. When collecting the seedlings, shoots, and roots, each sample should be harvested within one minute.
  2. Each sample should have three replicates at least.
  3. When adding salicylic acid-sulphate acid into the tube, the interval time between samples should be the same.
  4. When boiling the samples, the boiling time is at least 20 min.
  5. The cuvettes used for measuring the OD410 of the samples are quartz cuvettes.


  1. 500 mg/L (0.0357 mol/L) KNO3 standard solution
    0.7221 g KNO3 is dissolved in deionized water, and then add dH2O up to 200 ml
    Store at 4 °C
  2. 5% (w/v) salicylic acid-sulphuric acid
    5 g salicylic acid in 100 ml sulphuric acid
    Protect from light, store at 4 °C and use within 7 days
  3. 8% (w/v) NaOH solution
    80 g NaOH in 1 L distilled water
    Store in a glass bottle with rubber stopper


This research was supported by NSFC grant (31170230) and Taishan Scholar Foundation to Y. W. This protocol was mainly based on the method of Cataldo et al. (1975) and Vendrell et al. (1990).


  1. Bremner, J. M. (1965). Methods of soil analysis. Part 2. In: Black, C. A. (Ed.). ASA pp: 1216-1219.
  2. Carlson, R. M. and Keeney, D. R. (1971). Specific ion electrodes: Techniques and uses in soil, plant, and water analysis. In: Walsh, L. M. (Ed.). Instrumental Methods for Analysis of Soils and Plant Tissue. Soil Sci Soc Am pp: 39-65.
  3. Cataldo, D. A., Maroon, M., Schrader, L. E. and Youngs, V. L. (1975). Rapid colorimetric determination of nitrate in plant-tissue by nitration a salicylic-acid. Commun Soil Sci Plan 6: 71-80.
  4. Huffman, S. A. and Barbarick, K. A. (1981). Solid nitrate analysis by cadmium reduction. Comm Soil Sci Pl Anal 12:79-89.
  5. Vendrell, P. F. and Zupancic, J. (1990). Determination of soil nitrate by transnitration of salicylic-acid. Commun Soil Sci Plan 21: 1705-1713.
  6. Xu, N., Wang, R., Zhao, L., Zhang, C., Li, Z., Lei, Z., Liu, F., Guan, P., Chu, Z., Crawford, N. M. and Wang, Y. (2016). The Arabidopsis NRG2 protein mediates nitrate signaling and interacts with and regulates key nitrate regulators. Plant Cell 28(2): 485-504.


氮是植物生长必需的大量营养素,植物中的硝酸盐含量可以反映土壤的氮素供应。 在这里,我们提供了水杨酸方法来评估植物组织中的硝酸盐含量。 该方法可靠稳定,可作为植物组织中硝酸盐测定的良好选择。
【背景】氮是植物正常生长发育所需的重要营养元素。通常大多数植物主要以有氧条件下生长的硝酸盐形式吸收氮(Xu et al。,2016)。为了确定植物中的硝酸盐积累,我们需要测试植物不同组织中的硝酸盐含量。有一些测定硝酸盐的方法,例如电位法(Carlson and Keeney,1971),苯并二磺酸法(Bremner,1965),还原还原(Huffman and Barbarick,1981)等方法。这些方法具有一些缺点,例如敏感性较低,干扰,技术人员接触致癌物质(Cataldo等,1975; Vendrell和Zupancic,1990)
在这里,我们提供没有干扰,可靠和稳定的水杨酸方法。硝酸水杨酸在高度酸性条件下通过硝酸盐和水杨酸的反应形成。在碱性(pH> 12)条件下,该复合物为黄色,在410nm具有最大吸收。吸光度与硝酸盐含量成正比。因此,组织中的硝酸盐含量可以根据其吸光度计算。该方法适用于测定植物中的硝酸盐浓度。

关键字:硝酸盐含量, 植物, 水杨酸 - 硫酸, NaOH, 标准曲线, 消煮, OD410


  1. 1.5 ml Eppendof管
  2. 12ml塑料培养管(Greiner Bio One,目录号:184261)
  3. 石英比色皿
  4. 根系和/或芽(7日龄幼苗)
  5. 硝酸钾(KNO 3)(国药化学试剂,目录号:10017218)
  6. 去离子水
  7. MS媒体
  8. 液氮
  9. 水杨酸(国药化学试剂,目录号:30163517)
  10. 硫酸(98%)(国药化学试剂,目录号:100216008)
  11. 氢氧化钠(NaOH)(国药化学试剂,目录号:10019718)
  12. 500mg / L(0.0357mol / L)KNO 3标准溶液(参见食谱)
  13. 5%(w / v)水杨酸 - 硫酸(参见食谱)
  14. 8%(w / v)NaOH溶液(参见食谱)


  1. 50ml烧瓶
  2. 冷冻混合球磨机(RETCH,型号:MM400)
  3. 可见光分光光度计(PGENERAL,目录号:T6)
  4. 离心机(Eppendorf,型号:5424)


  1. 高强


  1. 标准曲线
    1. 为了制成标准曲线,加入1ml,2ml,3ml,4ml,6ml,8ml,10ml和12ml的NO 3标准溶液( 500mg / L)分别转移到八个50ml烧瓶中,并将去离子水加入每个溶液中以使总体积达到50ml。一系列标准溶液的浓度分别为10,20,30,40,60,80,100和120 mg / L。 10,20,30,40,60,80,100和120mg / L KNO 3的摩尔浓度为0.0007,0.0014,0.0021,0.0029,0.0043,0.0057,0.0071,0.0086摩尔/升, L。
    2. 将0.1ml各标准溶液分别转移到12ml管中。使用0.1ml去离子水作为对照。
    3. 向每个管中加入0.4ml水杨酸 - 硫酸并充分混合,然后在室温下温育所有反应20分钟。
    4. 向每个管中加入9.5毫升8%(w / v)NaOH溶液,冷却管(由反应产生的热量)至室温(约20-30分钟),并测量OD 410 / sub>值与控制参考。
    5. 绘制硝酸盐浓度为水平轴的标准曲线,吸光度为垂直轴。然后,可以根据标准曲线获得回归方程(图1)。具体方法如下:
      1. 打开一个Excel,输入A列中的OD <410>数值和列B中标准溶液的硝酸盐浓度。选择所有包含值的单元格,然后插入散点图。
      2. 选择图中的任意数据点,右键单击,选择添加趋势线,选择线性和显示方程,然后获得标准曲线和回归方程。

      图1.标准曲线。 使用10,20,30,40,60,80,100和120mg / L标准溶液建立标准曲线。误差条代表生物重复的SD(n = 4)。根据标准曲线,回归方程为C(μg/ ml)= 140.86×OD 410 < - 1.1831,其中C代表硝酸盐浓度。

  2. 拟南芥中的硝酸盐测定
    1. 将幼苗在半MS培养基上生长7天(如图2所示),并分别收集幼苗,芽和根以测定硝酸盐含量。

      图2.拟南芥幼苗的水培栽培系统 A.阿拉伯种子 种子种植在纱布网上已经通过高压灭菌消毒的250微米的筛孔尺寸)。 B.纱布网放在支架上。确保烧杯中的中等水平达到纱布网。

    2. 将每个称重的样品(≤0.1g,例如,在半MS上生长的约20-25个7天龄的野生型幼苗)通过液氮在1.5ml管中冷冻,并将每个样品研磨成粉末,频率为30 /秒使用RETCH MM400 1分钟。
    3. 在管中加入1ml去离子水,并在100℃下煮沸20分钟(至少)。
    4. 将样品以15,871×g离心10分钟,并将0.1ml上清液转移到新的12-ml管中。使用0.1ml去离子水作为对照。
    5. 向每个管中加入0.4ml水杨酸 - 硫酸,将样品充分混合,然后在室温下孵育反应20分钟。
    6. 向每个管中加入9.5毫升8%(w / v)NaOH溶液,并将管冷却至室温(约20-30分钟)。用参考的控件测量每个样品的OD值410 值。
    7. 根据上述步骤中得到的OD 410值,用回归方程式计算硝酸盐浓度(℃)C(μg/ ml)= 140.86×OD 410 <在步骤A(图1)中获得的1.1831。
    8. 使用以下公式计算硝酸盐含量:
      Y = CV / W
      Y:硝酸盐含量(μg/ g),
      C:用OD 410计算的硝酸盐浓度作为步骤B7(μg/ ml)回归方程式,


表1. WT的根的硝酸盐含量将幼苗在半MS培养基上生长7天,并收集根以测定硝酸盐。

注意:植物组织中硝酸盐含量的其他结果发表在“拟南芥NRG2蛋白介导硝酸盐信号并与之相互作用并调节关键的硝酸盐调节剂”( http://www.plantcell.org/content/28/2/485 。长 )。


  1. 收集幼苗,芽和根时,每个样品应在一分钟内收获
  2. 每个样本应至少有三个重复。
  3. 当向管中加入水杨酸 - 硫酸酸时,样品间隔时间应相同
  4. 煮沸样品时,煮沸时间至少为20分钟
  5. 用于测量样品的OD 410的比色皿是石英比色杯。


  1. 500mg / L(0.0357mol / L)KNO 3标准溶液
    将0.7221g KNO 3溶于去离子水中,然后加入dH 2 O 2至200ml
  2. 5%(w / v)水杨酸 - 硫酸
    5克水杨酸在100ml硫酸中的溶液 防止光照,储存在4°C,并在7天内使用
  3. 8%(w / v)NaOH溶液
    80 g NaOH在1 L蒸馏水中 存放在带有橡胶塞的玻璃瓶中


这项研究得到了国家自然科学基金资助项目(31170230)和泰山学者基金会对Y. W.的支持。该方案主要基于Cataldo等人(1975)和Vendrell等人的方法。 (1990)。


  1. Bremner,JM(1965)。&nbsp; 土壤方法分析。第2部分。在In:Black,C.A。(Ed。)。 pp:1216-1219。
  2. Carlson,RM和Keeney,DR(1971)。&lt; a class =“ke-insertfile”href =“https://dl.sciencesocieties.org/publications/books/abstracts/acsesspublicati/instrumentalmet/39”target =“ _blank“>特定离子电极:土壤,植物和水分析中的技术和用途。在Walsh,LM(Ed。)。土壤和植物组织分析的工具方法。土壤科学研究院 pp:39-65。
  3. Cataldo,DA,Maroon,M.,Schrader,LE and Youngs,VL(1975)。&lt; a class =“ke-insertfile”href =“http://www.tandfonline.com/doi/abs/10.1080/ 00103627509366547“target =”_ blank“>通过硝化水杨酸来快速比色测定植物组织中的硝酸盐。通用土壤科学计划 6:71-80。
  4. Huffman,SA和Barbarick,KA(1981)。&nbsp; 坚实通过镉还原进行硝酸盐分析。通用土壤科学分析12:79-89。
  5. Vendrell,PF和Zupancic,J.(1990)。&lt; a class =“ke-insertfile”href =“http://www.tandfonline.com/doi/abs/10.1080/00103629009368334?journalCode=lcss20”target = “_blank”>通过水杨酸的硝化来测定土壤硝酸盐。通用土壤科学计划 21:1705-1713。
  6. Xu,N.,Wang,R.,Zhao,L.,Zhang,C.,Li,Z.,Lei,Z.,Liu,F.,Guan,P.,Chu,Z.,Crawford,NM and Wang ,Y.(2016)。拟南芥NRG2蛋白介导硝酸盐信号传导并与重要的硝酸盐调节剂相互作用和调控。植物细胞28(2):485-504。
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引用:Zhao, L. and Wang, Y. (2017). Nitrate Assay for Plant Tissues. Bio-protocol 7(2): e2029. DOI: 10.21769/BioProtoc.2029.