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Detection of Nitric Oxide and Determination of Nitrite Concentrations in Arabidopsis thaliana and Azospirilum brasilense
拟南芥和巴西固氮螺菌中一氧化氮的检测和硝酸盐浓度的测定

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Abstract

There is now general agreement that nitric oxide (NO) is an important and almost ubiquitous signal in plants. Nevertheless, there are still many controversial observations and different opinions on the importance and functions of NO in plants. Partly, this may be due to the difficulties in detecting and even more in quantifying NO. Here, we summarize protocols for detecting NO and quantifying nitrite concentration in Arabidopisis seedlings and for the NO real time measurement in biofilms formed by the plant growth promoting rhizobacteria Azospirillum brasilense (A. brasilense). NO in oxygen-containing aqueous solution has a short half-life that is often attributed to a rapid oxidation to nitrite. Here we detail the use of the fluorescent probe DAF-FM DA and the electrochemical method for directly detecting and quantifying NO, respectively, and the Griess reagent to indirectly detect NO through its oxidized nitrite form. These protocols could be useful in a variety of cell types and different tissues of plants, and for microorganisms.

Keywords: Nitric oxide(一氧化氮), Nitrite(亚硝酸盐), Plants(植物), Rhizosphere microorganisms(根际微生物)

Part I. In vitro determination of nitrite concentration

Materials and Reagents

  1. Square Petri dishes (Deltalab, catalog number: 200204 )
  2. Multi-well plates (96 well) (Deltalab, catalog number: 900010 )
  3. 10-day-old Arabidopsis ecotype Columbia Col-0
  4. Murashige and Skoog Basal Salt Mixture (MS) (Sigma-Aldrich, catalog number: M5524 )
  5. Sulfanilamide (Sigma-Aldrich, catalog number: S9251 )
    Note: Working solution 1% (w/v) Sulfanilamide in 5% (v/v) phosphoric acid. Storage at 4 ºC in dark.
  6. N-(1-Naphthyl)ethylenediamine dihydrochloride (NED) (Sigma-Aldrich, catalog number: 33461 )
    Note: Working solution 0.1% (w/v) NED in H2O. Storage at 4 ºC in dark.
  7. Standard nitrite solution (Sigma-Aldrich, catalog number: 237213 )
    Note: Working solution 100 µM sodium nitrite in Mili Q water.
  8. Sodium phosphate dibasic (Sigma-Aldrich, catalog number: S0876 )
  9. Sodium phosphate monobasic (Sigma-Aldrich, catalog number: 0751 )
  10. Buffer A (100 mM phosphate buffer, pH 7.4) (see Recipes)

Equipment

  1. Centrifuge (Thermo Fisher Scientific, model: Sorvall Legend Micro 17R )
  2. Elisa read plate (Metrolab 980 microplate reader)

Procedure

  1. Grow Arabidopsis in Petri dishes containing ½ strength MS medium for 5 d and then transfer to treatment (100 mM NaCl) for 5 d or more (step 1, Figure 1).
  2. Ground 100 mg of seedlings or dissect the plant into root and shoot to a powder under liquid nitrogen in a mortar and suspend samples in 300 µl of 100 mM sodium phosphate (pH 7.4) [step 2(a), Figure 1].
  3. Centrifuge samples at 10,000 x g for 15 min at 4 °C [step 2(b), Figure 1b].
  4. Use the supernatant for nitrite and protein quantification: For nitrite, load 50 µl of supernatant in a well of the Elisa plate by triplicated [step 2(c), Figure 1]. For protein using Bradford (1976), take 1 or 2 µl of supernatant by triplicated.
  5. For nitrite Standard, prepare 1 ml of a 100 μM nitrite solution [step 2(c), Figure 1]. Dispense 50 μl of the Buffer A into the wells in rows B-H. Add 100 μl of the 100 μM nitrite solution to the remaining 3 wells in the first row. Immediately perform 6 serial two-fold dilutions (50 μl/well) in triplicate down the plate to generate the Nitrite Standard reference curve (100, 50, 25, 12.5, 6.25, 3.13 and 1.56 μM). Do not add any nitrite solution to the last set of wells (0 μM) as this will serve as the blank measurement.
  6. Allow the Sulfanilamide Solution and NED Solution to equilibrate to room temperature (15-30 min). Dispense 50 μl of the Sulfanilamide Solution to all experimental samples and wells containing the dilution series for the Nitrite Standard reference curve [step 2(c), Figure 1].
  7. Incubate 5-10 min at room temperature, protected from light.
  8. Dispense 50 μl of the NED Solution to all wells (step 3, Figure 1).
  9. Incubate at room temperature for 5-10 min, protected from light. A purple/magenta color will begin to form immediately, when the Griess reaction occurred (step 3, Figure 1).
  10. Measure absorbance in a plate reader with a filter between 520 nm and 550 nm, a single measure for per well is sufficient. The measurement should be done within 30 min after step 9 (step 3, Figure 1). Color may fade after this time.
  11. Use the standard curve to calculate the nitrite concentration in the samples. Refer the resulting data as nitrite per µg of protein. Use the equation (step 4, Figure 1) for nitrite calculation, replace the valor of “y” for the absorbance detected for the sample and then calculated the “x” valor, this valor correspond a nitrite concentration.


    Figure 1. Nitrite determination by Griess assay in Arabidopsis seedlings

Recipes

  1. Buffer A (100 mM phosphate buffer, pH 7.4)
    77.4 mM Sodium phosphate dibasic
    22.6 mM Sodium phosphate monobasic
    Storage at room temperature

Part II. In vivo detection of NO

Method 1. Electrochemical detection of NO

Materials and Reagents

  1. Multi-well plates (24 wells flat bottom) (Sigma-Aldrich, Corning® Costar®, catalog number: CLS3527 )
  2. 20 ml borosilicate glass vial (Thermo Fisher Scientific, catalog number: 033377 )
  3. Azospirillum brasilense Sp245 strain
  4. Standard nitrite solution (Sigma-Aldrich, catalog number: 237213 )
    Note: Working solution 100 µM sodium nitrite in Mili Q water. Prepare freshly for use.
  5. Potassium iodide (Sigma-Aldrich, catalog number: 746428 )
  6. Sulfuric acid (Merck Millipore Corporation, catalog number: 100732 )
  7. DL-Malic acid (Sigma-Aldrich, catalog number: 240176 )
  8. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  9. Magnesium sulfate heptahydrate (MgSO4.7H2O) (Sigma-Aldrich, catalog number: 230391 )
  10. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  11. Calcium chloride hydrate (CaCl2.H2O) (Sigma-Aldrich, catalog number: 202940 )
  12. Ethylenediaminetetraacetic acid ferric sodium salt (Fe-EDTA) (Sigma-Aldrich, catalog number: E6760 )
  13. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P1767 )
  14. Potassium nitrate (KNO3) (Sigma-Aldrich, catalog number: P8291 )
  15. Sodium molybdate dehydrate (NaMoO4.2H2O) (Sigma-Aldrich, catalog number: 331058 )
  16. Manganese(II) sulfate monohydrate (MnSO4) (Sigma-Aldrich, catalog number: M7634 )
  17. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
  18. Copper(II) sulfate pentahydrate (CuSO4.5H2O) (Sigma-Aldrich, catalog number: C8027 )
  19. Zinc sulfate heptahydrate (ZnSO4.7H2O) (Sigma-Aldrich, catalog number: Z0251 )
  20. Phosphate buffered saline, pH 7.4 (Sigma-Aldrich, catalog number: P4417 )
  21. Calibration solution (see Recipes)
    Note: Prepare freshly for use.
  22. Buffer B (see Recipes)
  23. NFb-malic medium (see Recipes)

Equipment

  1. Nitric Oxide Measuring System (NOMS) (e.g. Innovative Instruments Inc., model: inNO-T-II System )
  2. NO-specific sensor (e.g. Innovative Instruments Inc., model: amiNO-2000 )
  3. Sensoready (Innovative Instrument) device
    Sensor calibration
    1. Before calibrating the sensor should be polarized for a few hours, preferably overnight immersed in calibration solution or Mili Q water and connected to Sensoready device.
    2. Turn on PC and open NOMS software.
    3. The sensor is calibrated by a chemical reaction for NO production based on the conversion of nitrite to NO in acidic solution in the presence of iodide ion. The reaction has a molar ratio 1:1, meaning that the amount of NO produced equals the amount of nitrite added. In this protocol we used the term “NO/nitrite” concentration to unify both.
      Note: Other methods such as using the NO donor (±)-S-Nitroso-N-acetylpenicillamine (SNAP) and NO saturated solutions can be assayed (Allen et al., 2003).
    4. Immerse the tip of the sensor in the calibration solution. Zero the background using Zero button from NOMS software.
    5. Add 10 µl of nitrite standard solution to a 20 ml calibration solution while stirring. Wait until the current reaches its maximum potential and begins to decline.
    6. Zero the background again pressing Zero button from NOMS software.
    7. Repeat steps 5-6 at least three more times with adding 20, 40 and 80 µl of nitrite standard solution, respectively.
    8. Measure the peak height of each addition on NOMS software by placing cursor on peak (panel A, Figure 2). Plot current (pA) vs. concentration of NO/nitrite (nM) to make a reference curve (panel B, Figure 2).
      Note: The final volume in the vial is 20 ml, and the NO concentration range is 0-400 nM. 


    Figure 2. Construction of standard curve (A, B) and determination of NO in A. brasilense Sp245 biofilm sample (C) using a NO electrode

Software

  1. NOMS software

Procedure

  1. Grow A. brasilense Sp245 (2 ml per well) on tissue plates for 2 d in NFb-malic medium under static conditions to allow biofilm formation.
  2. Immediately before use, stabilize the microelectrode for 15 min running in Buffer B followed by 15 min in NFb-malic medium.
  3. Zero the background.
  4. Immerse microelectrode 3-4 mm in the bacterial culture and start recording changes on current potential. Usually, 30-40 min recording time is needed per sample to measure NO production in Azospirillum static cultures.
  5. Enter the obtained current value in the standard curve to establish NO concentration of the samples. Use the equation (panel B, Figure 2) to transform the current values to concentration of NO, replace the value of “y” for the pA detected for the sample and then calculated the “x” value, this value correspond to NO concentration in nM.
    Note: The concentration of nitrite in a sample can be measured in vitro by injecting a certain volume of the sample (bacterial culture supernatants) into an acid/iodide solution in which nitrite is converted to NO and then detected by the sensor.

Recipes

  1. Calibration solution
    Weigh and dissolve 20 mg of potassium iodide in 15 ml of Mili Q water and 2 ml of 1 M sulfuric acid. After potassium iodine dissolved completely, add Mili Q water to make 20 ml of solution.
    Note: Once this solution becomes light yellow, due to the formation of iodine in the solution, discard and prepare a new solution.
  2. Buffer B
    PBS was prepared according to product specifications. One tablet of PBS dissolved in 200 ml of deionized water yields 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride (pH 7.4), at 25 °C. Storage at room temperature.
  3. NFb-malic medium [modified as in Arruebarrena et al. (2013)]
    For 1 L NFb-malic medium pH 6.5 with nitrate as N source: 3.7 g Malic acid, 5 ml K2HPO4 10% (w/v), 2 ml MgSO4.7H2O 10% (w/v), 1 ml NaCl 10% (w/v), 2 ml CaCl2.H2O 1% (w/v), 2 ml Micronutrients solution, 4 ml Fe-EDTA 1.64% (w/v), 4.5 g KOH, 1.39 g KNO3.
    For 200 ml of micronutrients solution: 200 mg NaMoO4.2H2O, 235 mg MnSO4, 280 mg H3BO3, 8 mg CuSO4.5H2O, 24 mg ZnSO4.7H2O.


Method 2.
NO Fluorometric assay

Materials and Reagents

  1. Square Petri dishes (Deltalab, catalog number: 200204 )
  2. Multi-well plates (12 or 24 wells) (Biofil®)
  3. Microscopic glass slides (Deltalab, catalog number: D100001 ) and cover slips (Deltalab, catalog number: D102440 )
  4. Five-day-old Arabidopsis root seedlings ecotype Columbia Col-0
  5. Murashige and Skoog Basal Salt Mixture (MS) (Sigma-Aldrich, catalog number: M5524 )
  6. 1 mM Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 449709 )
    Note: Storage at room temperature.
  7. 0.25 mM KCl (Sigma-Aldrich, catalog number: P3911 )
    Note: Storage at room temperature.
  8. DAF-FM diacetate (DAF-FM DA) (Thermo Fisher Scientific, Molecular probesTM, catalog number: D-23844 )
    Note: Storage at -20 ºC.
  9. Abscisic acid (Sigma-Aldrich, catalog number: A1049 )
    Note: Storage at -20 ºC.
  10. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  11. 5 mM MES buffer (pH 5.7) (Sigma-Aldrich, catalog number: M3671 )
  12. DAF-FM diacetate (DAF-FM DA) stock solution (see Recipes)
  13. Buffer C (see Recipes)

Equipment

  1. Bright field and fluorescent microscope Eclipse E200 microscope (Nikon Corporation) (http://www.nikon.com/)

Procedure

  1. Grow Arabidopsis in Petri dishes in ½ strength MS medium for 4 to 5 d. Take the seedlings with tweezer and transfer to microplate wells containing abscisic acid (ABA) 10 µM in ½ strength liquid MS medium for 2 h.
  2. Replace the ABA solution with 1 ml Buffer C containing 10 µM DAF FM DA (step 1, Figure 3).
  3. Incubate seedlings at room temperature protected from light for 20 min followed by washing with 1 ml of fresh Buffer C for 20 min.
  4. Mount seedlings on glass slides and cover with cover slips. Samples are visualized under bright field and epifluorescent microscopy (excitation 490 nm; emission 525 nm) (step 2, Figure 3).


    Figure 3. Nitric Oxide detection by DAF-FA DA in Arabidopsis root seedlings

Recipes

  1. DAF-FM diacetate (DAF-FM DA) stock solution
    5 mM DAF-FM DA in dimethyl sulfoxide (DMSO)
  2. Buffer C
    Storage at room temperature
    5 mM MES buffer, adjusted with KOH at pH 5.7
    1 mM CaCl2
    0.25 mM KCl

Acknowledgments

This research was supported by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT: PICTs 2383/2011 and 2621/2011 to L.L. and N.C.-A., respectively), the Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 0903/2011 to L. L.), and institutional grants from the Universidad Nacional de Mar del Plata, Argentina. This protocol was adapted from the published work (Palma et al., 2013; Foresi et al., 2015).

References

  1. Allen, B. W. and Piantadosi, C. A. (2003). Electrochemical activation of electrodes for amperometric detection of nitric oxide. Nitric Oxide 8(4): 243-252.
  2. Arruebarrena Di Palma, A., Pereyra, C. M., Moreno Ramirez, L., Xiqui Vazquez, M. L., Baca, B. E., Pereyra, M. A., Lamattina, L. and Creus, C. M. (2013). Denitrification-derived nitric oxide modulates biofilm formation in Azospirillum brasilense. FEMS Microbiol Lett 338(1): 77-85.
  3. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  4. Coneski, P. N. and Schoenfisch, M. H. (2012). Nitric oxide release: part III. Measurement and reporting. Chem Soc Rev 41(10): 3753-3758.
  5. Foresi, N., Mayta, M. L., Lodeyro, A. F., Scuffi, D., Correa-Aragunde, N., Garcia-Mata, C., Casalongue, C., Carrillo, N. and Lamattina, L. (2015). Expression of the tetrahydrofolate-dependent nitric oxide synthase from the green alga Ostreococcus tauri increases tolerance to abiotic stresses and influences stomatal development in Arabidopsis. Plant J 82(5): 806-821.

简介

现在普遍认为一氧化氮(NO)是植物中重要的和几乎普遍存在的信号。然而,对于植物中NO的重要性和功能仍有许多有争议的观察和不同的观点。部分地,这可能是由于检测中的困难以及甚至更多的在定量NO。在这里,我们总结了在拟南芥幼苗中检测NO和定量亚硝酸盐浓度的方案以及在由植物生长促进根瘤菌(Azospirillum brasilense)形成的生物膜中的NO实时测量( > A。brasilense )。含氧水溶液中的NO具有短的半衰期,其通常归因于快速氧化成亚硝酸盐。在这里我们详细的使用荧光探针DAF-FM DA和电化学方法分别直接检测和定量NO,Griess试剂通过其氧化的亚硝酸盐形式间接检测NO。这些方案可用于多种细胞类型和植物的不同组织以及微生物。

关键字:一氧化氮, 亚硝酸盐, 植物, 根际微生物

第I部分。体外亚硝酸盐浓度测定

材料和试剂

  1. 方形培养皿(Deltalab,目录号:200204)
  2. 多孔板(96孔)(Deltalab,目录号:900010)
  3. 10天的拟南芥生态型Columbia Col-0
  4. Murashige和Skoog基础盐混合物(MS)(Sigma-Aldrich,目录号:M5524)
  5. 磺胺(Sigma-Aldrich,目录号:S9251)
    注意:工作溶液1%(w/v)磺胺在5%(v/v)磷酸中。储存于4℃,黑暗中。
  6. N-(1-萘基)乙二胺二盐酸盐(NED)(Sigma-Aldrich,目录号:33461)
    注意:在H 2 中工作溶液为0.1%(w /储存于4℃,黑暗中。
  7. 标准亚硝酸盐溶液(Sigma-Aldrich,目录号:237213)
    注意:在Mili Q水中使用100μM亚硝酸钠。
  8. 磷酸氢二钠(Sigma-Aldrich,目录号:S0876)
  9. 磷酸二氢钠(Sigma-Aldrich,目录号:0751)
  10. 缓冲液A(100mM磷酸盐缓冲液,pH7.4)(参见配方)

设备

  1. 离心机(Thermo Fisher Scientific,型号:Sorvall Legend Micro 17R)
  2. Elisa读板(Metrolab 980酶标仪)

程序

  1. 在含有1/2强度MS培养基的培养皿中生长5天,然后转移至处理(100mM NaCl)5天或更长时间(步骤1,图1),生长拟南芥。
  2. 在液氮中,在研钵中研磨100mg秧苗或将植物切成根并将其切成粉末,并将样品悬浮在300μl100mM磷酸钠(pH 7.4)中[步骤2(a),图1]。
  3. 在4℃下以10000×g离心样品15分钟[步骤2(b),图1b]。
  4. 使用上清液进行亚硝酸盐和蛋白质定量:对于亚硝酸盐,通过三次重复[在步骤2(c),图1]在Elisa板的孔中加载50μl上清液。对于使用Bradford(1976)的蛋白质,取1或2μl上清液一式三份
  5. 对于亚硝酸盐标准品,制备1ml的100μM亚硝酸盐溶液[步骤2(c),图1]。将50μl缓冲液A分配到B-H行的孔中。添加100微升100微米亚硝酸盐溶液到第一行的其余3口井。立即进行6次连续两倍稀释(50μl/孔),一式三份地向下平板,以产生亚硝酸盐标准参考曲线(100,50,25,12.5,6.25,3.13和1.56μM)。不要向最后一组孔(0μM)中添加任何亚硝酸盐溶液,因为这将用作空白测量。
  6. 使磺胺酰胺溶液和NED溶液平衡至室温(15-30分钟)。向所有实验样品和含有亚硝酸盐标准参考曲线[步骤2(c),图1]的稀释系列的孔中加入50μl磺胺酰胺溶液。
  7. 在室温下孵育5-10分钟,避光保护
  8. 向所有孔中分配50μlNED溶液(步骤3,图1)。
  9. 在室温下孵育5-10分钟,避光保存。当发生Griess反应时,紫色/品红色将立即开始形成(图1的步骤3)。
  10. 在具有在520nm和550nm之间的滤光片的读板仪中测量吸光度,每孔的单次测量是足够的。测量应在步骤9(步骤3,图1)后30分钟内完成。此后,颜色可能会褪色。
  11. 使用标准曲线计算样品中的亚硝酸盐浓度。将所得数据作为亚硝酸盐/μg蛋白质。使用方程(步骤4,图1)进行亚硝酸盐计算,将样品的吸光度代入"y",然后计算"x"值,该值对应亚硝酸盐浓度。

    图1.通过在拟南芥苗中的Griess测定法亚硝酸盐的测定

食谱

  1. 缓冲液A(100mM磷酸盐缓冲液,pH7.4) 77.4mM磷酸氢二钠 22.6mM磷酸二氢钠 室温下贮存

第二部分。 检测到NO

方法1 电化学检测NO

材料和试剂

  1. 多孔板(24孔平底)(Sigma-Aldrich,Corning公司,目录号:CLS3527)
  2. 20ml硼硅酸盐玻璃小瓶(Thermo Fisher Scientific,目录号:033377)

  3. Sp245菌株
  4. 标准亚硝酸盐溶液(Sigma-Aldrich,目录号:237213)
    注意:工作溶液在Mili Q水中100μM亚硝酸钠。请立即准备使用。
  5. 碘化钾(Sigma-Aldrich,目录号:746428)
  6. 硫酸(Merck Millipore Corporation,目录号:100732)
  7. DL-苹果酸(Sigma-Aldrich,目录号:240176)
  8. 磷酸氢二钾(K 2 HPO 4)(Sigma-Aldrich,目录号:P3786)
  9. 硫酸镁七水合物(MgSO 4)7H 2 O(Sigma-Aldrich,目录号:230391)
  10. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  11. 氯化钙水合物(CaCl 2)(Sigma-Aldrich,目录号:202940)。
  12. 乙二胺四乙酸铁钠盐(Fe-EDTA)(Sigma-Aldrich,目录号:E6760)
  13. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P1767)
  14. 硝酸钾(KNO 3)(Sigma-Aldrich,目录号:P8291)
  15. 将脱水钼酸钠(NaMoO 4 2H 2 O)(Sigma-Aldrich,目录号:331058)
  16. 硫酸锰(II)一水合物(MnSO 4)(Sigma-Aldrich,目录号:M7634)
  17. 硼酸(H 3 BO 3)(Sigma-Aldrich,目录号:B6768)
  18. 硫酸铜(II)五水合物(CuSO 4·5H 2 O·5H 2 O)(Sigma-Aldrich,目录号:C8027)
  19. 硫酸锌七水合物(ZnSO 4·7H 2 O 7H 2 O)(Sigma-Aldrich,目录号:Z0251)
  20. 磷酸盐缓冲盐水,pH 7.4(Sigma-Aldrich,目录号:P4417)
  21. 校准溶液(参见配方)
    注意:请立即准备使用。
  22. 缓冲液B(参见配方)
  23. NFb-苹果酸培养基(参见食谱)

设备

  1. 一氧化氮测量系统(NOMS)(例如,Innovative Instruments Inc.,型号:NO-T-II系统)
  2. NO特异性传感器(例如,Innovative Instruments Inc.,型号:amiNO-2000)
  3. Sensoready(创新仪器)装置
    传感器校准
    1. 在校准之前,传感器应该极化几个小时, 优选在校准溶液或Mili Q水中浸泡过夜 并连接到Sensoready设备。
    2. 打开PC并打开NOMS软件。
    3. 传感器通过用于NO生成的化学反应校准 基于亚硝酸盐在酸性溶液中的NO转化率 存在碘离子。该反应具有1:1的摩尔比,意味着 ?NO产生的量等于亚硝酸盐的加入量。在这里 协议,我们使用术语"NO /亚硝酸盐"浓度来统一两者 注意:其他方法,如使用NO供体 (±)-S-亚硝基-N-乙酰青霉胺(SNAP)和NO饱和溶液 可以测定(Allen等,2003)。
    4. 将传感器的尖端浸入校准溶液中。使用NOMS软件的Zero按钮将背景调零。
    5. 加入10微升亚硝酸盐标准溶液到20毫升校准溶液 同时搅拌。等待直到电流达到其最大电位 ?开始下降。
    6. 从NOMS软件再次按下零按钮将背景调零。
    7. 重复步骤5-6至少三次,分别加入20,40和80μl的亚硝酸盐标准溶液。
    8. 通过放置测量NOMS软件上每次添加的峰高 光标在峰(图A,图2)。绘制电流(pA)对浓度 的NO /亚硝酸盐(nM)作为参考曲线(图B,图2) 注意:小瓶中的最终体积为20ml,NO浓度范围为0-400nM。


    图2.使用NO电极构建标准曲线(A,B)和确定NO在巴西杆菌中的NO。Sp245生物膜样品(C)

软件

  1. NOMS软件

程序

  1. 在静止条件下在NFb-苹果酸培养基中在组织平板上生长2×2个Sp245(每孔2ml)以允许生物膜形成。
  2. 在使用前,将微电极在缓冲液B中稳定15分钟,然后在NFb-苹果酸培养基中稳定15分钟。
  3. 将背景调零。
  4. 浸泡微细电极3-4毫米的细菌培养和开始记录电流电位的变化。通常,每个样品需要30-40分钟的记录时间,以测量在azospirillum 静态培养物中的NO产生。
  5. 在标准曲线中输入获得的当前值,以确定样品的NO浓度。使用方程(图2中的图B)将电流值转换为NO的浓度,用样品检测的pA代替"y"的值,然后计算"x"值,该值对应于NO浓度nM。
    注意:样品中亚硝酸盐的浓度可以通过将一定体积的样品(细菌培养物上清液)注入酸/碘化物溶液中在体外测量,其中亚硝酸盐转化为NO,然后通过传感器检测。

食谱

  1. 校准溶液
    称量并溶解20mg碘化钾在15ml Mili Q水和2ml 1M硫酸中。待碘钾完全溶解后,加入Mili Q水制成20ml溶液。
    注意:一旦溶液变成浅黄色,由于溶液中形成碘,请丢弃并准备新的溶液。
  2. 缓冲区B
    根据产品规格制备PBS。在25℃下,一片溶于200ml去离子水中的PBS产生0.01M磷酸盐缓冲液,0.0027M氯化钾和0.137M氯化钠(pH7.4)。在室温下储存。
  3. NFb-苹果酸培养基[按Arruebarrena等人修改(2013)]
    对于使用硝酸盐作为N源的1L NFB-苹果酸培养基pH6.5:3.7g苹果酸,5ml K 2 HPO 4 10%(w/v),2ml MgSO 4·7H 2 O 10%(w/v),1ml NaCl 10%(w/v),2ml CaCl 2, 2ml微量营养素溶液,4ml Fe-EDTA 1.64%(w/v)水溶液,1ml(w/v) ,4.5g KOH,1.39g KNO 3。
    对于200ml微量营养素溶液:200mg NaMoO 4+,2H 2 O,235mg MnSO 4,280 mg H 3 BO 3,8mg CuSO 4 sub。,5 H 2 O 3, 24mg ZnSO 4 sub。 7H 2 O。


方法2 无荧光测定

材料和试剂

  1. 方形培养皿(Deltalab,目录号:200204)
  2. 多孔板(12或24孔)(Biofil )
  3. 显微镜载玻片(Deltalab,目录号:D100001)和盖玻片(Deltalab,目录号:D102440)
  4. 五天的拟南芥根幼苗生态型Columbia Col-0
  5. Murashige和Skoog基础盐混合物(MS)(Sigma-Aldrich,目录号:M5524)
  6. 1mM氯化钙(CaCl 2)(Sigma-Aldrich,目录号:449709)
    注意:在室温下储存。
  7. 0.25mM KCl(Sigma-Aldrich,目录号:P3911) 注意:在室温下储存。
  8. DAF-FM二乙酸酯(DAF-FM DA)(Thermo Fisher Scientific,Molecular probes TM ,目录号:D-23844) 注意:存放于-20oC。
  9. 脱落酸(Sigma-Aldrich,目录号:A1049)
    注意:存放于-20oC。
  10. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  11. 5mM MES缓冲液(pH 5.7)(Sigma-Aldrich,目录号:M3671)
  12. DAF-FM二乙酸酯(DAF-FM DA)储备溶液(参见配方)
  13. 缓冲区C(参见配方)

设备

  1. 明场和荧光显微镜Eclipse E200显微镜(Nikon Corporation)(http://www.nikon.com/)

程序

  1. 在半强度MS培养基中的培养皿中生长拟南芥 4至5天。取秧苗用镊子,并转移到含有脱落酸(ABA)10微米在1/2强度液体MS培养基的微孔板孔2小时。
  2. 用含有10μMDAF FM DA的1ml缓冲液C(步骤1,图3)替换ABA溶液
  3. 孵育幼苗在室温避光保存20分钟,然后用1ml新鲜的缓冲液C洗涤20分钟。
  4. 在玻璃幻灯片和盖子的登上幼木有盖子的。样品在明场和落射荧光显微镜(激发490nm;发射525nm)下可见(步骤2,图3)。


    图3. DAF-FA DA在拟南芥根苗中的一氧化氮检测

食谱

  1. DAF-FM二乙酸酯(DAF-FM DA)储液
    5mM DAF-FM DA在二甲基亚砜(DMSO)中的溶液
  2. 缓冲区C
    室温下贮存
    5mM MES缓冲液,用KOH在pH5.5调节 1mM CaCl 2
    0.25mM KCl

致谢

这项研究得到了Agencia Nacional dePromociónCientíficayTecnológica(ANPCyT:PICTs 2383/2011和2621/2011分别对LL和NC-A。),Consejo Nacional de InvestigacionesCientíficasyTécnicas(PIP 0903/2011 to LL ),以及来自阿根廷国家马德普拉塔大学的机构赠款。该方案改编自公开的工作(Palma等人,2013; Foresi等人,2015)。

参考文献

  1. Allen,B.W。和Piantadosi,C.A。(2003)。 电化学活化电极,用于电流检测一氧化氮。一氧化氮 8(4):243-252。
  2. Arruebarrena Di Palma,A.,Pereyra,C.M.,Moreno Ramirez,L.,Xiqui Vazquez,M.L.,Baca,B.E.,Pereyra,M.A。,Lamattina,L.and Creus, 反硝化衍生的一氧化氮可调节巴西半岛的生物膜形成。 FEMS Microbiol Lett 338(1):77-85。
  3. Bradford,M.M。(1976)。 利用蛋白质染料结合原理的快速灵敏的微克量蛋白定量方法。 Anal Biochem 72:248-254。
  4. Coneski,P.N。和Schoenfisch,M.H。(2012)。 一氧化氮释放:第三部分。 Measurement and reporting。 Chem Soc Rev 41(10):3753-3758。
  5. Foresi,N.,Mayta,M.L.,Lodeyro,A.F.,Scuffi,D.,Correa-Aragunde,N.,Garcia-Mata,C.,Casalongue,C.,Carrillo,N.and Lamattina, 来自绿藻Ostreococcus tauri的四氢叶酸依赖性一氧化氮合酶的表达增加对非生物胁迫的耐受性并影响拟南芥中的气孔发育。 。 82 82(5):806-821。
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引用:Foresi, N., Correa-Aragunde, N., Amenta, M., Arruebarrena, A., Creus, C. and Lamattina, L. (2016). Detection of Nitric Oxide and Determination of Nitrite Concentrations in Arabidopsis thaliana and Azospirilum brasilense. Bio-protocol 6(6): e1765. DOI: 10.21769/BioProtoc.1765.
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