| In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts
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Author:
Date:
2018-04-05
[Abstract] Nitrate reductase (NR) reduces the major plant nitrogen source, NO3-, into NO2-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude extracts. This protocol offers simple procedure and data analysis to compare NR activity of multiple samples.
[摘要] 硝酸还原酶（NR）将主要的植物氮源NO 3 N-2还原为NO 2 - 2。 NR活性可以通过其最终产物，亚硝酸盐在最佳条件下通过其吸光度来测量。 在这里，我们提供了一个详细的协议，用于测量来自拟南芥粗提物的NR的相对酶活性。 该协议提供简单的程序和数据分析来比较多个样品的NR活性。
【背景】氮是植物所需的主要营养素，主要以硝酸盐的形式吸收。硝酸还原酶是高等植物中首次同化氮的酶。植物硝酸还原酶的同型二聚体如下催化硝酸根的NAD（P）H依赖性还原为亚硝酸根：
NO <3> + NADH + H +→NO - + NAD + H 测量NR活性的方法可能是研究影响NR活性的生物因素的有力工具（Park等人，2011）。氮同化影响植物中氨基酸的含量，因此调节NR活性可用于提高某些作物的质量（Croy和Hageman，1970; Dalling和Loyn，1977; Ruan等人，1998） ）。在该协议中，在优化的缓冲液条件下限制时间内亚硝酸盐浓度增加作为可比值获得。亚硫酸盐浓度通过Griess测定法通过其在540nm处的吸光度来测量。简言之，亚硝酸盐与磺胺酸形成重氮盐，然后N-（1-萘基）乙二胺二盐酸盐形成有色偶氮化合物。可以比较这些值以确定样品如何具有不同的NR活性。此外，通过简单的过程可以将这些数值转换为精确增加的亚硝酸盐浓度。 ...
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| Detection of Nitric Oxide and Determination of Nitrite Concentrations in Arabidopsis thaliana and Azospirilum brasilense
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Author:
Date:
2016-03-20
[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 differing opinions on the importance and functions of NO in plants. Partly, this may be due to the difficulties in detecting and quantifying NO. Here, we summarize protocols for detecting NO and quantifying nitrite concentration in Arabidopsis seedlings. We also present a method to measure NO in biofilms formed by the plant growth promoting rhizobacteria Azospirillum brasilense (A. brasilense). NO in oxygen-containing aqueous solutions 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 ...
[摘要] 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。
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| Localisation and Quantification of Reactive Oxygen Species and Nitric Oxide in Arabidopsis Roots in Response to Fungal Infection
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Author:
Date:
2014-10-05
[Abstract] Nitric oxide and reactive oxygen species have emerged as important signalling molecules in plants. The half-lives of NO and ROS are very short therefore rapid and precise measurements are required for the understanding biological roles of these redox active species. Various organelles and compartments generate NO and ROS thus it is important to determine precise location of these free radicals in order to understand their signalling roles. Diaminofluorescen (DAF) and fluorescent 2', 7'-dichlorofluorescein (DCF) dyes are employed to determine NO and ROS localisation. The advantage of this approach is that the dyes diffuse precisely to NO and ROS producing sites and generate fluorescence which can be detected by fluorescence- or confocal laser scanning microscopes. However, this technique ...
[摘要] 一氧化氮和活性氧在植物中作为重要的信号分子出现。 NO和ROS的半衰期非常短，因此需要快速和精确的测量来理解这些氧化还原活性物质的生物学作用。各种细胞器和隔室产生NO和ROS，因此重要的是确定这些自由基的精确位置，以了解他们的信号传导作用。使用二氨基荧光素（DAF）和荧光2'，7'-二氯荧光素（DCF）染料来确定NO和ROS定位。这种方法的优点是染料精确扩散到NO和ROS产生位点，并产生可以通过荧光或共聚焦激光扫描显微镜检测的荧光。然而，这种技术有其缺点;特别是需要建立荧光信号的特异性。因此，需要诸如cPTIO和抗坏血酸的ROS的使用清除剂来确认荧光信号的特异性，并且理想地，由于与每种方法相关的优点和缺点，理想地确认使用其它方法获得的数据（Gupta和Igamberdiev，2013 ）。在这里我们描述了一种方法来检测响应于木霉属，镰刀菌属使用DAF，气相Griess试剂测定和DCF荧光方法的感染的拟南芥根的NO和ROS产生。
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