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
发布: 2017年05月20日第7卷第10期 DOI: 10.21769/BioProtoc.2280 浏览次数: 20882
评审: Dennis NürnbergZhanwu DaiAnonymous reviewer(s)
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The authors used this protocol in:
Nov 2016
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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.
Materials and Reagents
- 0.1-10 µl pipette tips (NIPPON Genetics, catalog number: 30470 )
- 1-200 µl pipette tips (NIPPON Genetics, catalog number: 30430 )
- 100-1,000 µl pipette tips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 111-N-Q )
- 2.0 ml microtube with O-ring (SARSTEDT, catalog number: 72.693 )
- 1.5 ml microtube (WATSON, catalog number: 131-5155C )
- Assay plate (Iwaki, catalog number: 3881-096 )
- Gloves and eye protection
- Arabidopsis thaliana ecotype Col-0
- Ultrapure water (Milli-Q, Millipore, 18 MΩ cm)
- Liquid N2
- Sulfuric acid (Wako Pure Chemical Industries, catalog number: 192-04696 )
- Salicylic acid (Wako Pure Chemical Industries, catalog number: 196-14861 )
- Sodium hydroxide (Wako Pure Chemical Industries, catalog number: 198-13765 )
- Potassium nitrate (Wako Pure Chemical Industries, catalog number: 160-04035 )
- Reaction reagent 1 (see Recipes)
- Reaction reagent 2 (see Recipes)
Equipment
- Pipettes (Nichiryo, model: Nichipet EX II )
- Heat block (TAITEC, model: DTU-1B )
- Refrigerated centrifuge (TOMY DIGITAL BIOLOGY, model: MX-300 )
- Vortex mixer (Scientific Industries, catalog number: SI-0236 )
- Multimode plate reader (PerkinElmer, model: EnSpire® 2300 )
Note: Versatile spectrophotometers are practicable. - Spectrophotometer (Shimadzu, model: UV-1650PC )
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
植物科学 > 植物新陈代谢 > 氮
细胞生物学 > 细胞新陈代谢 > 其它化合物
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