A critical comparison of two high-throughput ascorbate analyses methods for plant samples.
A critical comparison of two high-throughput ascorbate analyses methods for plant samples.
复制标题
DOI：
10.1016/j.plaphy.2013.06.006
复制
发表时间：
2013-09
期刊：
影响因子：
--
通讯作者：
Y. Ueda;Linbo Wu;M. Frei
中科院分区：
文献类型：
--
作者：
Y. Ueda;Linbo Wu;M. Frei
抗坏血酸（AsA）是参与植物逆境反应和发育的重要代谢物。因此，在植物科学的许多领域，包括高通量和关键应用中，有必要对AsA含量进行量化。在这项研究中，我们比较了两种不同的基于微孔板的AsA检测方法，它们适用于高通量应用：基于抗坏血酸氧化酶（AO）的检测方法和基于双吡啶（DPD）的检测方法。这些方法在关键应用中进行了比较，即(i) AsA浓度非常低时，如在外质体提取物中，（ii）当植物含有干扰光谱测量的色素时，以及（iii）当植物含有高铁浓度干扰颜色反应时。DPD法测量精度较高，内标AsA回收率较高。另一方面，AO法对低水平AsA更为敏感。这在测定水稻外胞体AsA浓度时具有优势，其浓度远低于整个组织。与DPD法相反，AO法还有一个优点，即植物色素和植物组织中的高铁浓度不会干扰分析。总之，两种检测方法都有各自的优点，选择合适的方法取决于具体的应用。
Ascorbate (AsA) is an important metabolite involved in stress response and development of plants. Therefore it is necessary to quantify the AsA content in many fields of plant science, including high throughput and critical applications. In this study we compared two different microplate-based AsA assays, which are suitable for high throughput applications: an ascorbate oxidase (AO)-based assay and a dipyridyl (DPD)-based assay. These methods were compared in critical applications, i.e. (i) when AsA concentrations were very low such as in apoplastic extracts, (ii) when plants contained pigments interfering with the spectrometric measurements, and (iii) when plants contained high iron concentration interfering with the color reactions. The precision of measurements was higher with the DPD method, as illustrated by higher recovery rates of internal AsA standards. On the other hand, the AO method was more sensitive to low levels of AsA. This was an advantage in determining apoplastic AsA concentration in rice, which was substantially lower than that of whole tissues. The AO method also had the advantage that plant pigments and high iron concentrations in plants tissues did not interfere with the analysis, as opposed to the DPD assay. In conclusion, both assays had advantages and the choice of a suitable method depends on the specific application.