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Simple Spectroscopic Determination of Nitrate, Nitrite, and Ammonium in Arabidopsis thaliana
拟南芥中硝酸盐、亚硝酸盐和铵的简单光谱测定   

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Abstract

Plants use nitrate, nitrite, and ammonium as inorganic nitrogen (N) sources. These N compounds are included in plant tissues at various concentrations depending on the balance between their uptake and assimilation. Thus, the contents of nitrate, nitrite, and ammonium are physiological indicators of plant N economy. Here, we describe a protocol for measurement of these inorganic N species in A. thaliana shoots or roots.

Keywords: Arabidopsis thaliana(拟南芥), Nitrate(硝酸盐), Nitrite(亚硝酸盐), Ammonium(铵)

Background

Determination of inorganic N content is important for predicting the ability of a plant to uptake and assimilate N. Researchers often use techniques requiring expensive equipment, such as high-performance liquid chromatography (HPLC), for these measurements. The present protocol is based on versatile spectrometry with cheap reagents, making it practicable for many researchers. Nitration of salicylic acid by nitrate occurs under acidic conditions, and subsequent addition of an alkaline solution results in a yellow complex. Under acidic conditions, nitrite reacts with sulfanilamide to produce a diazonium compound that undergoes diazocoupling with N-(1-naphthyl)ethylenediamine to form a pink azo compound. Ammonium can be determined as a blue indophenol derivative under the catalytic influence of a nitroprusside salt. Each experimental procedure is easy, rapid, and simple.

Part I. Determination of nitrate (NO3-)

Materials and Reagents

  1. 0.1-10 µl pipette tips (NIPPON Genetics, catalog number: 30470 )
  2. 1-200 µl pipette tips (NIPPON Genetics, catalog number: 30430 )
  3. 100-1,000 µl pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 111-N-Q )
  4. 2.0 ml microtube with O-ring (SARSTEDT, catalog number: 72.693 )
  5. 1.5 ml microtube (WATSON, catalog number: 131-5155C )
  6. Assay plate (Iwaki, catalog number: 3881-096 )
  7. Gloves and eye protection
  8. Arabidopsis thaliana ecotype Col-0
  9. Ultrapure water (Milli-Q, Millipore, 18 MΩ cm)
  10. Liquid N2
  11. Sulfuric acid (Wako Pure Chemical Industries, catalog number: 192-04696 )
  12. Salicylic acid (Wako Pure Chemical Industries, catalog number: 196-14861 )
  13. Sodium hydroxide (Wako Pure Chemical Industries, catalog number: 198-13765 )
  14. Potassium nitrate (Wako Pure Chemical Industries, catalog number: 160-04035 )
  15. Reaction reagent 1 (see Recipes)
  16. Reaction reagent 2 (see Recipes)

Equipment

  1. Pipettes (Nichiryo, model: Nichipet EX II )
  2. Heat block (TAITEC, model: DTU-1B )
  3. Refrigerated centrifuge (TOMY DIGITAL BIOLOGY, model: MX-300 )
  4. Vortex mixer (Scientific Industries, catalog number: SI-0236 )
  5. Multimode plate reader (PerkinElmer, model: EnSpire® 2300 )
    Note: Versatile spectrophotometers are practicable.
  6. Spectrophotometer (Shimadzu, model: UV-1650PC )

Procedure

  1. Extraction of nitrate from A. thaliana shoots or roots
    1. Excise and weigh fresh shoots or roots, put them into a 2 ml microtube with an O-ring, and freeze them in liquid N2.
      Note: Samples can be stored at -80 °C before use. We usually use 10-50 mg of samples (fresh weight) for nitrate measurements. Nitrate is extractable from the samples with or without being powdered in liquid N2 (Xu et al., 2016). We routinely use whole shoots or roots as they are for the extraction.
    2. Add 10 vols. of ultrapure water.
      Note: The volume of water added is determined based on the assumption that the pellet derived from 1 mg fresh weight has a volume of about 1 µl. Addition of water preheated at 80 °C denatures nitrate reductase more quickly, avoiding nitrate metabolism during extraction. The whole sample should be submerged in water.
    3. Incubate at 100 °C for 20 min with the lid closed.
      Note: The tubes should be shaken by hand or vortexed every 5 min for complete extraction.
    4. Centrifuge the cooled tubes at 20,400 x g for 10 min at room temperature. Use the supernatants for nitrate determination.
      Note: Supernatants can be stored at -80 °C before use.

  2. Determination of nitrate from A. thaliana shoots or roots
    1. Add 40 µl of reaction reagent 1 (see Recipes) to a 1.5 ml microtube to determine the apparent nitrate concentration.
    2. Add 40 µl of sulfuric acid instead of reaction reagent 1 to another 1.5 ml microtube to determine the nonspecific background concentration.
    3. Add 10 µl of supernatant.
      Note: Supernatants are often sticky. We usually attach the supernatant to the wall of the microtube.
    4. Spin down the contents and vortex them thoroughly.
    5. Incubate at room temperature for 20 min.
      Note: The incubation temperature is usually maintained at 20-25 °C.
    6. Gently add 1 ml of reaction reagent 2 (see Recipes), and vortex until the contents become clear.
      Note: This neutralization process generates heat. The obtained yellow product is stable for 48 h in the dark.
    7. Measure the absorbance at 410 nm using a spectrophotometer.

  3. Preparation of nitrate standard curve
    1. Prepare a nitrate dilution series using potassium nitrate (0, 0.125, 0.25, 0.5, 1, 2, 4, and 8 mM) in ultrapure water.
      Note: The dilution series can be stored at -20 °C.
    2. Add 40 µl of reaction reagent 1 to a 1.5 ml microtube.
    3. Add 10 µl of a standard solution.
    4. Spin down the contents and vortex them thoroughly.
    5. Incubate at room temperature for 20 min.
    6. Gently add 1 ml of reaction reagent 2, and vortex until the contents become clear.
    7. Measure the absorbance at 410 nm using the spectrophotometer and construct a standard curve (Figure 1). A picture of a representative nitrate dilution series is shown in Figure 2.


      Figure 1. Standard curve generated using the absorbance at 410 nm for known concentrations of nitrate


      Figure 2. Picture of a representative nitrate dilution series

Data analysis

  1. A typical nitrate standard curve is shown in Figure 1. From the curve, the nitrate concentration (mM) can be determined using the formula: (Abs410 - intercept)/slope.
  2. Calculate the apparent nitrate concentration (mM) of the supernatant using the standard curve.
  3. Calculate the nonspecific background concentration (mM) of the supernatant using the standard curve.
  4. Calculate the true nitrate concentration (mM) of the supernatant by subtracting the nonspecific value from the apparent value.
  5. Determine the nitrate content of the sample (µmol g-1 fresh weight) using the formula: [true nitrate concentration (mM)] x [extracted volume (ml)]/[fresh weight (g)].

Notes

Sulfuric acid, salicylic acid, and sodium hydroxide are very dangerous and harmful for humans and the environment. Wear gloves and eye protection, and discard waste liquid properly. Please make sure that RISK assessments are carried out before conducting experiments. At least two technical replicates should be obtained for each sample and standard. More than five biological replicates are desirable to obtain reliable data for statistical analysis. In the above experimental procedure, the determination is limited to the range of 1.25-80 µmol nitrate g-1 fresh weight. If a result exceeds this limit, the extract should be diluted appropriately using ultrapure water, depending on the nitrate concentration of the sample.

Recipes

  1. Reaction reagent 1
    0.05% (w/v) salicylic acid in sulfuric acid
    Prepare freshly each day and avoid exposure to light
  2. Reaction reagent 2
    8% (w/v) NaOH in ultrapure water
    Store at 4 °C in a polypropylene screw-cap tube with the lid tightly closed

Part II. Determination of nitrite (NO2-)

Materials and Reagents

  1. 0.1-10 µl pipette tips (NIPPON Genetics, catalog number: 30470 )
  2. 1-200 µl pipette tips (NIPPON Genetics, catalog number: 30430 )
  3. 100-1,000 µl pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 111-N-Q )
  4. 2.0 ml safe-lock tube (Eppendorf, catalog number: 0107-23-31-33 )
  5. 5 mm zirconia beads (BMS, catalog number: ZZ50-0001 )
  6. 1.5 ml microtube (WATSON, catalog number: 131-5155C )
  7. Assay plate (Iwaki, catalog number: 3881-096 )
  8. Gloves and eye protection
  9. Arabidopsis thaliana ecotype Col-0
  10. Liquid N2
  11. 1 mol/L hydrochloric acid (Nacalai Tesque, catalog number: 37314-15 )
  12. 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Dojindo, catalog number: 342-01375 )
  13. Ethylenediamine-N,N,N’,N’-tetraacetic acid (EDTA), disodium salt, dehydrate (Dojindo, catalog number: 345-01865 )
  14. L-Cysteine (Sigma-Aldrich, catalog number: 168149 )
  15. Sulfanilamide (Wako Pure Chemical Industries, catalog number: 191-04502 )
  16. N-1-Naphthylethylenediamine dihydrochloride (Wako Pure Chemical Industries, catalog number: 147-04141 )
  17. Ultrapure water (Milli-Q, Millipore, 18 MΩ cm)
  18. Extraction reagent 1 (see Recipes)
  19. Reaction reagent 3 (see Recipes)
  20. Reaction reagent 4 (see Recipes)

Equipment

  1. Pipettes (Nichiryo, model: Nichipet EX II )
  2. Sample disruptor with beads (QIAGEN, model: TissueLyser II )
    Note: Mortar and pestle are practicable.
  3. Refrigerated centrifuge (TOMY DIGITAL BIOLOGY, model: MX-300 )
  4. Vortex mixer (Scientific Industries, catalog number: SI-0236 )
  5. Multimode plate reader (PerkinElmer, model: EnSpire® 2300 )
    Note: Versatile spectrophotometers are practicable.
  6. Spectrophotometer (Shimadzu, model: UV-1650PC )

Procedure

  1. Extraction of nitrite from A. thaliana shoots or roots
    1. Excise and weigh fresh shoots or roots (approximately 50-100 mg fresh weight), put them into 2 ml safe-lock tubes, and freeze them in liquid N2.
      Note: We have not checked whether the frozen samples are storable or not. We recommend that the samples should be used for extraction and measurement of nitrite as soon as possible.
    2. Crush frozen samples into a fine powder with a sample disruptor using 5 mm zirconia beads (one bead per tube) or with a mortar and pestle under liquid N2 conditions.
    3. Add 5 vols. of extraction reagent 1 (see Recipes), vortex gently until the solution dissolves and becomes homogeneous, and keep the extracts on ice.
      Note: The volume of extraction buffer added is determined based on the assumption that the pellet derived from 1 mg fresh weight has a volume of about 1 µl.
    4. Centrifuge the extracts at 20,400 x g for 10 min at 4 °C. Use the supernatants for nitrite determination.
      Note: The supernatants can be used for measurements of nitrate reductase activity (Konishi and Yanagisawa, 2011).

  2. Determination of nitrite from A. thaliana shoots or roots
    1. Mix 150 µl of the supernatant with 150 µl of reaction reagent 3 (see Recipes) in a 1.5 ml microtube to determine the apparent nitrite concentration.
    2. Mix 150 µl of the supernatant with 150 µl of 1 mol/L hydrochloric acid instead of reaction reagent 3 to determine the nonspecific background concentration.
    3. Centrifuge the mixture at 20,400 x g for 10 min at 4 °C to remove precipitates.
    4. Mix 200 µl of the supernatant with 100 µl of reaction reagent 4 (see Recipes).
      Note: Supernatant:reagent 3:reagent 4 = 1:1:1 (v/v/v). If a larger volume is needed to measure the absorbance, this mixture can be scaled up.
    5. Incubate at room temperature for 15 min.
    6. Measure the absorbance at 540 nm using a spectrophotometer.

  3. Preparation of nitrite standard curve
    1. Prepare a nitrite dilution series using potassium nitrite (0, 5, 10, 20, and 40 µM) in the extraction reagent 1 (freshly prepared).
      Note: In most plants under normal growth conditions (except for those under anoxia/hypoxia or in which nitrite reduction is suppressed), little nitrite accumulates in the tissues. The range of the standard curve should be adjusted depending on the type of sample.
    2. Mix standard solutions with equal vols. of reaction reagent 3 and reaction reagent 4 in 1.5 ml microtubes.
    3. Incubate at room temperature for 15 min.
    4. Measure the absorbance at 540 nm using the spectrophotometer (Figure 3). A picture of a representative nitrite dilution series is shown in Figure 4.


      Figure 3. Standard curve generated using the absorbance at 540 nm for known concentrations of nitrite


      Figure 4. Picture of a representative nitrite dilution series

Data analysis

  1. A typical nitrite standard curve is shown in Figure 3. From the curve, the nitrite concentration (µM) can be determined using the formula: (Abs540 - intercept)/slope.
  2. Calculate the apparent nitrite concentration (µM) of the supernatant using the standard curve.
  3. Calculate the nonspecific background concentration (µM) of the supernatant using the standard curve.
  4. Calculate the true nitrite concentration (µM) of supernatant by subtracting the nonspecific value from the apparent value.
  5. Determine the nitrite content of the sample (nmol g-1 fresh weight) using the formula: [true nitrate concentration (µM)] x [extracted volume (ml)]/[fresh weight (g)].

Notes

Nitrite solutions are not very stable. Extraction and determination should be carried out as soon as possible. Hydrochloric acid is very dangerous and harmful to humans and the environment. Wear gloves and eye protection, and discard waste liquid properly. Please make sure that RISK assessments are carried out before conducting experiments. At least two technical replicates should be obtained for each sample and standard. More than five biological replicates are desirable to obtain reliable data for statistical analysis. In the above experimental procedure, the determination is limited to the range of 25-200 nmol nitrite g-1 fresh weight. If a result exceeds this limit, the extract should be diluted appropriately using extraction reagent 1, depending on the nitrite concentration of the sample. Nitrite is accumulated in tissues where nitrite reduction is inhibited under a limited supply of reducing equivalents (Hachiya et al., 2016).

Recipes

  1. Extraction reagent 1
    50 mM HEPES-KOH (pH 7.6)
    1 mM EDTA
    7 mM cysteine
    Note: Add cysteine just before use. Store solution without cysteine at 4 °C after autoclaving.
  2. Reaction reagent 3
    1% (w/v) sulfanilamide in 1 mol/L hydrochloric acid
    Prepare freshly each day
  3. Reaction reagent 4
    0.02% (w/v) N-1-naphthylethylenediamine dihydrochloride in ultrapure water
    Prepare freshly each day

Part III. Determination of ammonium (NH4+)

Materials and Reagents

  1. 0.1-10 µl pipette tips (NIPPON Genetics, catalog number: 30470 )
  2. 1-200 µl pipette tips (Nippon Genetics, catalog number: 30430 )
  3. 100-1000 µl pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 111-N-Q )
  4. 2.0 ml safe-lock tube (Eppendorf, catalog number: 0107-23-31-33 )
  5. 5 mm zirconia beads (BMS, catalog number: ZZ50-0001 )
  6. 1.5 ml microtube (WATSON, catalog number: 131-5155C )
  7. Gloves and eye protection
  8. Assay plate (Iwaki, catalog number: 3881-096 )
  9. Arabidopsis thaliana ecotype Col-0
  10. Ultrapure water (Milli-Q, Millipore, 18 MΩ cm)
  11. Liquid N2
  12. Chloroform (Wako Pure Chemical Industries, catalog number: 038-02606 )
  13. Acid-washed activated charcoal (Wako Pure Chemical Industries, catalog number: 035-18081 )
  14. 1 mol/L hydrochloric acid (Nacalai Tesque, catalog number: 37314-15 )
  15. Ammonia test kit (Ammonia Test Wako, Wako Pure Chemical Industries, catalog number: 277-14401 )
    Notes:
    1. This inexpensive kit ensures reliable measurement of ammonium based on modifications of the Okuda-Fujii method (Okuda and Fujii, 1965).
    2. The ammonia test kit is ready-to-use and can be stored at 4 °C for one and a half years. Instead of using the kit, it is possible to prepare the reaction reagents using commercial reagents based on the methods of Bräutigam et al. (2007).
  16. Extraction reagent 2 (see Recipes)

Equipment

  1. Pipettes (Nichiryo, model: Nichipet EX II )
  2. Sample disruptor with beads (QIAGEN, model: TissueLyser II )
    Note: Mortar and pestle are practicable.
  3. Refrigerated centrifuge (TOMY DIGITAL BIOLOGY, model: MX-300 )
  4. Heat block (TAITEC, model: DTU-1B )
  5. Vortex mixer (Scientific Industries, catalog number: SI-0236 )
  6. Microtube mixer (TOMY DIGITAL BIOLOGY, model: MT-360 )
  7. Multimode plate reader (PerkinElmer, model: EnSpire® 2300 )
    Note: Versatile spectrophotometers are practicable.
  8. Spectrophotometer (Shimadzu, model: UV-1650PC )

Procedure

  1. Extraction of ammonium from A. thaliana shoots or roots
    1. Excise and weigh fresh shoots or roots (approximately 10-100 mg fresh weight), put them into 2 ml safe-lock tubes, and freeze them in liquid N2.
      Note: Samples can be stored at -80 °C before use.
    2. Crush frozen samples into a fine powder with a sample disruptor using 5 mm zirconia beads (one bead per tube) or with a mortar and pestle under liquid N2 conditions.
    3. Add 1 ml of extraction reagent 2 (see Recipes), and vortex gently until the solution dissolves and becomes homogeneous.
    4. Add 500 µl of chloroform.
    5. Vortex the mixture gently at 4 °C for 15 min.
    6. Centrifuge the extracts at 12,000 x g for 10 min at 8 °C.
    7. Transfer the aqueous phase into a 1.5 ml microtube containing approximately 50 mg of acid-washed activated charcoal and then vortex the mixture.
    8. Centrifuge the extracts at 20,400 x g for 5 min at 8 °C. Use the supernatants for ammonium determination.
      Note: A series of purification steps eliminates interfering compounds for ammonium determination.

  2. Determination of ammonium from A. thaliana shoots or roots
    1. Mix 200 µl of the supernatant with 800 µl of the deproteinization reagent in the ammonia test kit in a 1.5 ml microtube and vortex.
      Note: The deproteinization reagent contains sodium tungstate and phosphate salt.
    2. Centrifuge the mixture at 2,300 x g for 5 min at 4 °C.
    3. Mix 200 µl of the supernatant with 200 µl of reagent A in the ammonia test kit and vortex.
      Note: Reagent A contains sodium nitroprusside and phenol.
    4. Add 100 µl of reagent B in the ammonia test kit and vortex.
      Note: Reagent B contains potassium hydroxide.
    5. Add 200 µl of reagent C in the ammonia test kit and vortex.
      Note: Reagent C contains sodium hypochlorite and potassium carbonate.
    6. Incubate at 37 °C for 20 min.
    7. Measure the absorbance at 630 nm using a spectrophotometer.
      Note: If the ammonium concentration exceeds the range of the standard dilution series, dilute the supernatant with the extraction reagent 2. In general, ammonium accumulates at higher levels in plant tissues grown under ammonium-containing conditions.

  3. Preparation of ammonium standard curve
    1. Prepare an ammonium dilution series using ammonium chloride (0, 12.5, 25, 50, 100, 200, and 400 µM) in the extraction reagent 2 (freshly prepared).
      Note: Ammonia Test Wako includes solutions for the preparation of the dilution series. However, their background composition resembles deproteinized blood because this kit is designed to measure ammonium in blood. Thus, the dilution series should be prepared with the extraction reagent 3.
    2. Mix 200 µl of the standard solution with 800 µl of the deproteinization reagent in the ammonia test kit in a 1.5 ml microtube and vortex.
      Note: The deproteinization reagent contains sodium tungstate and phosphate salt.
    3. Centrifuge the mixture at 800 x g for 5 min at 4 °C.
    4. Mix 200 µl of the supernatant with 200 µl of reagent A in the ammonia test kit and vortex.
    5. Add 100 µl of reagent B in the ammonia test kit and vortex.
    6. Add 200 µl of reagent C in the ammonia test kit and vortex.
    7. Incubate at 37 °C for 20 min.
    8. Measure the absorbance at 630 nm using the spectrophotometer (Figure 5). A picture of a representative ammonium dilution series is shown in Figure 6.


      Figure 5. Standard curve generated using the absorbance at 630 nm for known concentrations of ammonium


      Figure 6. Picture of a representative ammonium dilution series

Data analysis

  1. A typical ammonium standard curve is shown in Figure 5. From the curve, the ammonium concentration (µM) in the supernatant can be determined using the formula: (Abs630 - intercept)/slope.
  2. Determine the ammonium content of the sample (µmol g-1 fresh weight) using the formula: [ammonium concentration (µM)] x [extracted volume (ml)]/[fresh weight (g)]/1,000.

Notes

Chloroform should be used in a fume hood. Hydrochloric acid is very dangerous and harmful to humans and the environment. Wear gloves and eye protection, and discard waste liquid properly. Acid-washed activated charcoal should be used carefully in a fume hood to ensure that dust is not scattered. Please make sure that RISK assessments are carried out before conducting experiments. At least two technical replicates should be obtained for each sample and standard. More than five biological replicates are desirable to obtain reliable data for statistical analysis. In the above experimental procedure, a linear standard curve is not always achieved below 12.5 µM or above 400 µM. If a result exceeds this limit, the extract should be diluted appropriately using extraction reagent 2, depending on the ammonium concentration of the sample. High ammonium contents are accumulated in plants grown under ammonium at millimolar concentrations, but little ammonium is accumulated under nitrate conditions (Hachiya et al., 2012).

Recipes

  1. Extraction reagent 2
    0.1 mol/L hydrochloric acid

Acknowledgments

The present procedures are derived from the work of Cataldo et al. (1975), Rockel et al. (2002), Bräutigam et al. (2007), Konishi and Yanagisawa (2011), and Hachiya et al. (2012 and 2016). This work was supported by Building of Consortia for the Development of Human Resources in Science and Technology and by Core Research for Evolutional Science and Technology, Japan Science and Technology Agency.

References

  1. Bräutigam, A., Gagneul, D. and Weber, A. P. M. (2007). High-throughput colorimetric method for the parallel assay of glyoxylic acid and ammonium in a single extract. Anal Biochem 362(1): 151-153.
  2. Cataldo, D. A., Haroon, M., Schrader, L. E. and Youngs, V. L. (1975). Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun Soil Sci Plant Anal 6: 71-80.
  3. Hachiya, T., Ueda, N., Kitagawa, M., Hanke, G., Suzuki, A., Hase, T. and Sakakibara, H. (2016). Arabidopsis root-type ferredoxin:NADP(H) oxidoreductase 2 is involved in detoxification of nitrite in roots. Plant Cell Physiol 57(11): 2440-2450.
  4. Hachiya, T., Watanabe, C. K., Fujimoto, M., Ishikawa, T., Takahara, K., Kawai-Yamada, M., Uchimiya, H., Uesono, Y., Terashima, I. and Noguchi, K. (2012). Nitrate addition alleviates ammonium toxicity without lessening ammonium accumulation, organic acid depletion and inorganic cation depletion in Arabidopsis thaliana shoots. Plant Cell Physiol 53(3): 577-591.
  5. Konishi, M. and Yanagisawa, S. (2011). The regulatory region controlling the nitrate-responsive expression of a nitrate reductase gene, NIA1, in Arabidopsis. Plant Cell Physiol 52(5): 824-836.
  6. Okuda, H. and Fujii, S. (1966). Determination of blood ammonia by the spectrophotometric method. Saishin Igaku 21: 622-627.
  7. Rockel, P., Strube, F., Rockel, A., Wildt, J. and Kaiser, W. M. (2002). Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro. J Exp Bot 53(366): 103-110.
  8. 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.

简介

植物使用硝酸盐,亚硝酸盐和铵作为无机氮(N)源。这些N化合物根据其吸收和同化之间的平衡以各种浓度包含在植物组织中。因此,硝酸盐,亚硝酸盐和铵的含量是植物N经济的生理指标。在这里,我们描述了在A中测量这些无机N物质的方案。 thaliana 芽或根。

背景 无机氮含量的测定对于预测植物吸收和吸收氮的能力很重要。研究人员经常使用需要昂贵设备的技术,如高效液相色谱(HPLC),用于这些测量。本协议基于通用光谱法与廉价的试剂,使其适用于许多研究人员。在酸性条件下,硝酸盐引起水杨酸的硝化,随后加入碱性溶液会导致黄色复合物。在酸性条件下,亚硝酸盐与磺胺类反应产生重氮化合物,其与N - (1-萘基)乙二胺进行重氮偶联,形成粉红色偶氮化合物。在硝普钠盐的催化作用下,铵可以测定为蓝色靛酚衍生物。每个实验程序容易,快速,简单。

关键字:拟南芥, 硝酸盐, 亚硝酸盐, 铵

第一部分硝酸盐的测定(NO <3> sup>)

材料和试剂

  1. 0.1-10μl移液器吸头(NIPPON Genetics,目录号:30470)
  2. 1-200μl移液器吸头(NIPPON Genetics,目录号:30430)
  3. 100-1,000μl移液器吸头(Thermo Fisher Scientific,Thermo Scientific TM,目录号:111-N-Q)
  4. 2.0毫升带O型圈的微管(SARSTEDT,目录号:72.693)
  5. 1.5毫升微管(WATSON,目录号:131-5155C)
  6. 分析板(岩崎,目录号:3881-096)
  7. 手套和眼睛护理
  8. 拟南芥生态型Col-0
  9. 超纯水(Milli-Q,Millipore,18MΩcm)
  10. 液体N 2
  11. 硫酸(Wako Pure Chemical Industries,目录号:192-04696)
  12. 水杨酸(Wako Pure Chemical Industries,目录号:196-14861)
  13. 氢氧化钠(和光纯药,目录号:198-13765)
  14. 硝酸钾(Wako Pure Chemical Industries,目录号:160-04035)
  15. 反应试剂1(参见食谱)
  16. 反应试剂2(见配方)

设备

  1. 移液器(Nichiryo,型号:Nichipet EX II)
  2. 热块(TAITEC,型号:DTU-1B)
  3. 冷冻离心机(TOMY DIGITAL BIOLOGY,型号:MX-300)
  4. 涡旋搅拌机(Scientific Industries,目录号:SI-0236)
  5. 多模板读卡器(PerkinElmer,型号:EnSpire ® 2300)
    注意:多功能分光光度计是可行的。
  6. 分光光度计(Shimadzu,型号:UV-1650PC)

程序

  1. 从拟南芥芽或根中提取硝酸盐
    1. 消毒并称重新鲜的芽或根,将它们放入具有O形环的2ml微管中,并将其冷冻在液体N 2中。
      注意:样品可以储存在-80°C使用前。我们通常使用10-50毫克样品(鲜重)进行硝酸盐测量。硝酸盐可以从液体N 2中被粉碎的样品中提取出来(Xu等,2016)。我们常常使用整枝或根茎进行提取。
    2. 加10卷超纯水。
      注意:添加水的体积是基于假定来自1mg鲜重的淀粉的体积约为1μl。加入80℃预热的水更快地变性硝酸还原酶,避免硝酸盐在提取过程中的代谢。整个样品应浸没在水中。
    3. 在100°C孵育20分钟,盖子关闭。
      注意:应每5分钟用手摇动或旋转管,以完全提取。
    4. 将冷却的管在20,400×g下在室温下离心10分钟。使用上清液进行硝酸盐测定。
      注意:上清液可以储存在-80°C使用前。

  2. 从A测定硝酸盐。 thaliana 芽或根
    1. 加入40μl反应试剂1(参见食谱)至1.5 ml微量管,以确定硝酸盐的浓度
    2. 将40μl硫酸代替反应试剂1加入1.5ml微量管中,以确定非特异性背景浓度。
    3. 加入10μl上清液 注意:上清液常常粘稠。我们通常将上清液附着在微管的壁上。
    4. 旋转内容并彻底旋转它们。
    5. 在室温下孵育20分钟 注意:培养温度通常保持在20-25℃。
    6. 轻轻加入1ml反应试剂2(参见食谱),并涡旋直至内容物澄清 注意:这种中和过程产生热量。获得的黄色产品在黑暗中稳定48小时。
    7. 使用分光光度计测量410 nm处的吸光度
  3. 制备硝酸盐标准曲线
    1. 在超纯水中使用硝酸钾(0,0.125,0.25,0.5,1,2,4和8mM)制备硝酸盐稀释系列。
      注意:稀释系列可以储存在-20°C。
    2. 加入40μl反应试剂1至1.5 ml微管
    3. 加入10μl标准溶液
    4. 旋转内容并彻底旋转它们。
    5. 在室温下孵育20分钟
    6. 轻轻加入1ml反应试剂2,涡旋直至内容物澄清
    7. 使用分光光度计测量410nm处的吸光度并构建标准曲线(图1)。代表硝酸盐稀释系列的图片如图2所示

      图1.使用已知浓度的硝酸盐在410nm处的吸光度生成的标准曲线

      图2.代表性硝酸盐稀释系列的图片

数据分析

  1. 典型的硝酸盐标准曲线如图1所示。从曲线可以使用以下公式确定硝酸盐浓度(mM):(Abs 截距)/斜率。
  2. 使用标准曲线计算上清液的表观硝酸盐浓度(mM)。
  3. 使用标准曲线计算上清液的非特异性背景浓度(mM)
  4. 通过从表观值减去非特异性值计算上清液的真实硝酸盐浓度(mM)
  5. 使用以下公式确定样品的硝酸盐含量(μmolg -1)[真硝酸盐浓度(mM)]×[提取体积(ml)]/[鲜重(g)] 。

笔记

硫酸,水杨酸和氢氧化钠对人类和环境非常危险和有害。戴手套和眼睛保护,并妥善丢弃废液。在进行实验之前,请确保进行风险评估。每个样品和标准应至少进行两次技术复制。为了获得可靠的统计分析数据,需要五种以上的生物重复。在上述实验程序中,测定限于1.25-80μmol硝酸盐g -1重量的范围。如果结果超过此限制,则应根据样品的硝酸盐浓度,使用超纯水适当稀释萃取物。

食谱

  1. 反应试剂1
    0.05%(w/v)水杨酸在硫酸中 每天准备新鲜,避免暴露于光线
  2. 反应试剂2
    8%(w/v)NaOH在超纯水中
    储存于4°C的聚丙烯螺旋盖管中,盖子紧紧关闭
第二部分。亚硝酸盐的测定(NO 2 -

材料和试剂

  1. 0.1-10μl移液器吸头(NIPPON Genetics,目录号:30470)
  2. 1-200μl移液器吸头(NIPPON Genetics,目录号:30430)
  3. 100-1,000μl移液器吸头(Thermo Fisher Scientific,Thermo Scientific TM,目录号:111-N-Q)
  4. 2.0 ml安全锁管(Eppendorf,目录号:0107-23-31-33)
  5. 5毫米氧化锆珠(BMS,目录号:ZZ50-0001)
  6. 1.5毫升微管(WATSON,目录号:131-5155C)
  7. 分析板(岩崎,目录号:3881-096)
  8. 手套和眼睛护理
  9. 拟南芥生态型Col-0
  10. 液体N 2
  11. 1mol/L盐酸(Nacalai Tesque,目录号:37314-15)
  12. 2- [4-(2-羟基乙基)-1-哌嗪基]乙磺酸(HEPES)(Dojindo,目录号:342-01375)
  13. 乙二胺-N,N,N',N'-四乙酸(EDTA),二钠盐,脱水剂(Dojindo,目录号:345-01865)
  14. L-半胱氨酸(Sigma-Aldrich,目录号:168149)
  15. 磺胺(和光纯药,目录号:191-04502)
  16. <> -1-萘磺乙二胺二盐酸盐(和光纯药工业公司,目录号:147-04141)
  17. 超纯水(Milli-Q,Millipore,18MΩcm)
  18. 提取试剂1(参见食谱)
  19. 反应试剂3(见配方)
  20. 反应试剂4(参见食谱)

设备

  1. 移液器(Nichiryo,型号:Nichipet EX II)
  2. 带珠的样品破坏剂(QIAGEN,型号:TissueLyser II)
    注意:砂浆和杵是可行的。
  3. 冷冻离心机(TOMY DIGITAL BIOLOGY,型号:MX-300)
  4. 涡旋搅拌机(Scientific Industries,目录号:SI-0236)
  5. 多模板读卡器(PerkinElmer,型号:EnSpire ® 2300)
    注意:多功能分光光度计是可行的。
  6. 分光光度计(Shimadzu,型号:UV-1650PC)

程序

  1. 从A提取亚硝酸盐。 thaliana 芽或根
    1. 将新鲜的枝条或根(新鲜重量约50-100毫克)消毒并称重,将其放入2毫升安全锁管中,并将其冻结在液体N 2中。
      注意:我们尚未检查冻结样品是否可储存。我们建议尽快将样品用于提取和测量亚硝酸盐。
    2. 使用5毫米氧化锆珠(每管一个珠)或在液态N 2条件下用研钵和研杵将样品破碎机粉碎成细粉末。
    3. 加5卷的萃取剂1(参见食谱),轻轻旋涡直至溶液溶解并变得均匀,并将提取物保持在冰上。
      注意:添加的提取缓冲液的体积是基于来自1mg鲜重的淀粉的体积约为1μl的假设来确定的。
    4. 在40℃下将提取物离心10分钟,4℃。使用上清液进行亚硝酸盐测定。
      注意:上清液可用于测量硝酸还原酶活性(Konishi和Yanagisawa,2011)。

  2. 从A确定亚硝酸盐。 thaliana 芽或根
    1. 将150μl上清液与150μl反应试剂3(参见食谱)在1.5 ml微量管中混合,以确定表观亚硝酸盐浓度。
    2. 用150μl1 mol/L盐酸代替反应试剂3混合150μl上清液,以确定非特异性背景浓度。
    3. 在4℃下以20,400×g离心混合物10分钟以除去沉淀物。
    4. 将100μl上清液与100μl反应试剂4混合(参见食谱)。
      注意:上清液:试剂3:试剂4 = 1:1:1(v/v/v)。如果需要较大的体积来测量吸光度,则可以将该混合物放大。
    5. 在室温下孵育15分钟
    6. 使用分光光度计测量540 nm处的吸光度
  3. 制备亚硝酸盐标准曲线
    1. 在提取试剂1(新鲜制备的)中,使用亚硝酸钾(0,5,10,20和40μM)制备亚硝酸盐稀释系列。
      注意:在正常生长条件下的大多数植物中(缺氧/缺氧或抑制亚硝酸盐的除外),组织中积累了少量亚硝酸盐。标准曲线的范围应根据样品类型进行调整。
    2. 混合均等的标准溶液。的反应试剂3和反应试剂4在1.5ml微量管中
    3. 在室温下孵育15分钟
    4. 使用分光光度计测量540 nm处的吸光度(图3)。代表性亚硝酸盐稀释系列的图片如图4所示

      图3.使用已知亚硝酸盐浓度的540 nm处的吸光度生成的标准曲线


      图4.代表性亚硝酸盐稀释系列的图片

数据分析

  1. 典型的亚硝酸盐标准曲线如图3所示。从曲线可以使用以下公式确定亚硝酸盐浓度(μM):(Abs> 540 - 截距)/斜率。
  2. 使用标准曲线计算上清液的表观亚硝酸盐浓度(μM)
  3. 使用标准曲线计算上清液的非特异性背景浓度(μM)
  4. 通过从表观值中减去非特异性值,计算上清液的真实亚硝酸盐浓度(μM)
  5. 使用以下公式确定样品的亚硝酸盐含量(nmol g -1重量)[真硝酸盐浓度(μM)]×[提取体积(ml)]/[鲜重(g)] 。

笔记

亚硝酸盐溶液不太稳定。提取和确定应尽快进行。盐酸对人类和环境非常危险和有害。戴手套和眼睛保护,并妥善丢弃废液。在进行实验之前,请确保进行风险评估。每个样品和标准应至少进行两次技术复制。为了获得可靠的统计分析数据,需要五种以上的生物重复。在上述实验程序中,测定限于25-200nmol亚硝酸盐g -1 鲜重的范围。如果结果超过此限制,则应根据样品的亚硝酸盐浓度,使用萃取剂1适当稀释萃取液。亚硝酸盐在有限的还原当量供应量的情况下积累在亚硝酸盐还原被抑制的组织中(Hachiya等人,2016)。

食谱

  1. 提取试剂1
    50mM HEPES-KOH(pH 7.6)
    1 mM EDTA
    7 mM半胱氨酸
    注意:使用前加入半胱氨酸。高压灭菌后,在4℃下储存无半胱氨酸溶液。
  2. 反应试剂3
    1%(w/v)磺胺在1mol/L盐酸中的溶液 每天准备新鲜
  3. 反应试剂4
    在超纯水中0.02%(w/v)N 1 - 萘乙二胺二盐酸盐
    每天准备新鲜

第三部分。铵(NH 4 + )的测定

材料和试剂

  1. 0.1-10μl移液器吸头(NIPPON Genetics,目录号:30470)
  2. 1-200μl移液器吸头(Nippon Genetics,目录号:30430)
  3. 100-1000μl移液器吸头(Thermo Fisher Scientific,Thermo Scientific TM,目录号:111-N-Q)
  4. 2.0 ml安全锁管(Eppendorf,目录号:0107-23-31-33)
  5. 5毫米氧化锆珠(BMS,目录号:ZZ50-0001)
  6. 1.5毫升微管(WATSON,目录号:131-5155C)
  7. 手套和眼睛护理
  8. 分析板(岩崎,目录号:3881-096)
  9. 拟南芥生态型Col-0
  10. 超纯水(Milli-Q,Millipore,18MΩcm)
  11. 液体N 2
  12. 氯仿(和光纯药,目录号:038-02606)
  13. 酸洗活性炭(Wako Pure Chemical Industries,目录号:035-18081)
  14. 1mol/L盐酸(Nacalai Tesque,目录号:37314-15)
  15. 氨测试试剂盒(氨测试和光,和光纯药工业公司,目录号:277-14401)
    注意:
    1. 这种便宜的试剂盒基于Okuda-Fujii方法的改进(Okuda和Fujii,1965)确保了铵的可靠测量。
    2. 氨检测试剂盒即用即可,可在4℃保存一年半。代替使用试剂盒,可以使用基于Bräutigam等人的方法的商业试剂制备反应试剂。 (2007)。
  16. 提取试剂2(参见食谱)

设备

  1. 移液器(Nichiryo,型号:Nichipet EX II)
  2. 带珠的样品破坏剂(QIAGEN,型号:TissueLyser II)
    注意:砂浆和杵是可行的。
  3. 冷冻离心机(TOMY DIGITAL BIOLOGY,型号:MX-300)
  4. 热块(TAITEC,型号:DTU-1B)
  5. 涡旋搅拌机(Scientific Industries,目录号:SI-0236)
  6. 微管混合器(TOMY DIGITAL BIOLOGY,型号:MT-360)
  7. 多模板读卡器(PerkinElmer,型号:EnSpire ® 2300)
    注意:多功能分光光度计是可行的。
  8. 分光光度计(Shimadzu,型号:UV-1650PC)

程序

  1. 从A提取铵。 thaliana 芽或根
    1. 将新鲜的枝条或根(大约10-100毫克鲜重)重新粉碎并称重,将其放入2毫升安全锁管中,并将其冻结在液体N 2中。
      注意:样品可以在-80°C保存后再使用。
    2. 使用5毫米氧化锆珠(每管一个珠)或在液态N 2条件下用研钵和研杵将样品破碎机粉碎成细粉末。
    3. 加入1ml提取试剂2(参见食谱),轻轻涡旋,直至溶液溶解并变得均匀
    4. 加入500μl氯仿。
    5. 在4℃下轻轻旋转混合物15分钟
    6. 在12℃下将提取物离心10分钟,8℃
    7. 将水相转移到含有约50mg酸洗活性炭的1.5ml微量管中,然后涡旋混合物。
    8. 将提取物在20,400 x g处于8℃离心5分钟。使用上清液进行铵测定。
      注意:一系列纯化步骤消除了用于铵测定的干扰化合物。

  2. 从A测定铵。 thaliana 芽或根
    1. 将200μl上清液与氨测试试剂盒中的800μl去蛋白试剂混合在1.5ml微量管中并涡旋。
      注意:脱蛋白试剂含有钨酸钠和磷酸盐。
    2. 在4℃下以2,300×g离心混合物5分钟。
    3. 在氨测试试剂盒中将200μl上清液与200μl试剂A混合并涡旋 注意:试剂A含有硝普钠和苯酚。
    4. 在氨测试试剂盒中加入100μl试剂B并涡旋 注意:试剂B含有氢氧化钾。
    5. 在氨测试试剂盒中加入200μl试剂C并涡旋 注意:试剂C含有次氯酸钠和碳酸钾。
    6. 在37℃孵育20分钟。
    7. 使用分光光度计测量630 nm处的吸光度 注意:如果铵浓度超过标准稀释系列的范围,则用提取试剂2稀释上清液。通常,在含铵条件下生长的植物组织中,铵积累较高。

  3. 铵标准曲线的制备
    1. 在提取试剂2(新鲜制备)中,使用氯化铵(0,12.5,25,50,100,200和400μM)制备铵稀释系列。
      注意:氨测试Wako包括稀释系列的制备方法。然而,它们的背景组成类似于脱蛋白血液,因为该试剂盒旨在测量血液中的铵。因此,稀释系列应用提取试剂3制备。
    2. 将200μl标准溶液与800μl脱蛋白试剂在氨测试试剂盒中,在1.5 ml微量管中混合,并旋涡。
      注意:脱蛋白试剂含有钨酸钠和磷酸盐。
    3. 在800℃下将混合物在4℃下离心5分钟。
    4. 在氨测试试剂盒中将200μl上清液与200μl试剂A混合并涡旋
    5. 在氨测试试剂盒中加入100μl试剂B并涡旋
    6. 在氨测试试剂盒中加入200μl试剂C并涡旋
    7. 在37℃孵育20分钟。
    8. 使用分光光度计测量630 nm处的吸光度(图5)。代表性铵稀释系列的图片如图6所示

      图5.使用已知浓度的铵的630 nm处的吸光度生成的标准曲线

      图6. 代表性铵稀释系列的图片

数据分析

  1. 典型的铵标准曲线如图5所示。从曲线上可以用下列公式确定上清液中的铵浓度(μM):(Abs> 630截距)/斜率。 >
  2. 用[铵浓度(μM)]×[提取体积(ml)]/[鲜重(g)]//确定样品的铵含量(μmolg 1,000。

笔记

通风柜应使用氯仿。盐酸对人类和环境非常危险和有害。戴手套和眼睛保护,并妥善丢弃废液。在通风柜中应小心使用酸洗活性炭,以确保灰尘不会散落。在进行实验之前,请确保进行风险评估。每个样品和标准应至少进行两次技术复制。为了获得可靠的统计分析数据,需要五种以上的生物重复。在上述实验过程中,线性标准曲线并不总是在低于12.5μM或高于400μM的情况下实现。如果结果超过此限制,应根据样品的铵浓度,使用萃取剂2适当稀释萃取液。高铵含量在以毫摩尔浓度在铵下生长的植物中积累,但在硝酸盐条件下几乎不积聚铵(Hachiya等人,2012)。

食谱

  1. 提取试剂2
    0.1mol/L盐酸

致谢

本程序来自于Cataldo等人的工作。 (1975),Rockel等人(2002),Bräutigam等人(2007),Konishi和Yanagisawa(2011)和Hachiya等人(2012和2016)。这项工作得到了日本科技厅科技人力资源开发联盟和科技兴业核心研究的支持。

参考

  1. Bräutigam,A.,Gagneul D.和Weber,APM(2007)。用于单次提取物中乙醛酸和铵的平行测定的高通量比色法.Anal Biochem 362(1):151-153。
  2. Cataldo,DA,Haroon,M.,Schrader,LE and Youngs,VL(1975)。< a class ="ke-insertfile"href ="https://www.researchgate.net/publication/249072446_Rapid_Colorimetric_Determination_of_Nitrate_in_Plant-Tissue_by_Nitration_of_Salicylic-酸"目标="_ blank">通过水杨酸硝化快速比色测定植物组织中的硝酸盐。通用土壤科学植物分析 6:71-80。
  3. Hachiya,T.,Ueda,N.,Kitagawa,M.,Hanke,G.,Suzuki,A.,Hase,T。和Sakakibara,H。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27615794"target ="_ blank"> 拟南芥根型铁氧还蛋白:NADP(H)氧化还原酶2涉及解毒根系中的亚硝酸盐。植物细胞生理学57(11):2440-2450。
  4. Hachiya,T.,Watanabe,CK,Fujimoto,M.,Ishikawa,T.,Takahara,K.,Kawai-Yamada,M.,Uchimiya,H.,Uesono,Y.,Terashima,I.and Noguchi,K. (2012)。硝酸盐添加减轻铵毒性而不减少铵积累,有机酸消耗和拟南芥芽中的无机阳离子消耗。植物细胞生理学53(3):577-591。
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引用:Hachiya, T. and Okamoto, Y. (2017). Simple Spectroscopic Determination of Nitrate, Nitrite, and Ammonium in Arabidopsis thaliana. Bio-protocol 7(10): e2280. DOI: 10.21769/BioProtoc.2280.
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Zhe Ji
University of Oxford
Hi,
I have been trying to determine Arabidopsis leaf tissue nitrite (NO2-) with your protocol. I used 100 mg of wild type leaf tissue and followed each step but have got no color development at the end of the experiment. The standard curve worked fine so it's not likely due to the color development reagent. Could you please suggest a reason for this, and what I could do to improve?
Best wishes,
Luke
6/14/2017 3:24:01 AM Reply
Takushi Hachiya
Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences,, Nagoya University, Japan

Dear Dr. Luke,

Tissue nitrite concentration is often very low under normal growth condition in wild-type plants, because toxic nitrite is rapidly converted into ammonium by NiR. A significant detection may be made in the plants grown under hypoxia/anoxia or other nitrite-accumulating/containing conditions or in the mutants showing lower nitrite reduction.

6/15/2017 7:03:10 PM