Plant Biological Sciences Graduate Program, Department of Horticultural Science, and Microbial and Plant Genomics Institute, University of Minnesota, 1970 Folwell Avenue, Saint Paul, MN, 55108, USA.
Plant Methods. 2012 Aug 10;8(1):31. doi: 10.1186/1746-4811-8-31.
The plant hormone auxin, indole-3-acetic acid (IAA), plays important roles in plant growth and development. The signaling response to IAA is largely dependent on the local concentration of IAA, and this concentration is regulated by multiple mechanisms in plants. Therefore, the precise quantification of local IAA concentration provides insights into the regulation of IAA and its biological roles. Meanwhile, pathways and genes involved in IAA biosynthesis are not fully understood, so it is necessary to analyze the production of IAA at the metabolite level for unbiased studies of IAA biosynthesis.
We have developed high-throughput methods to quantify plant endogenous IAA and its biosynthetic precursors including indole, tryptophan, indole-3-pyruvic acid (IPyA), and indole-3-butyric acid (IBA). The protocol starts with homogenizing plant tissues with stable-labeled internal standards added, followed by analyte purification using solid phase extraction (SPE) tips and analyte derivatization. The derivatized analytes are finally analyzed by selected reaction monitoring on a gas chromatograph-mass spectrometer (GC-MS/MS) to determine the precise abundance of analytes. The amount of plant tissue required for the assay is small (typically 2-10 mg fresh weight), and the use of SPE tips is simple and convenient, which allows preparation of large sets of samples within reasonable time periods.
The SPE tips and GC-MS/MS based method enables high-throughput and accurate quantification of IAA and its biosynthetic precursors from minute plant tissue samples. The protocol can be used for measurement of these endogenous compounds using isotope dilution, and it can also be applied to analyze IAA biosynthesis and biosynthetic pathways using stable isotope labeling. The method will potentially advance knowledge of the role and regulation of IAA.
植物激素生长素（吲哚乙酸，IAA）在植物生长发育中起着重要作用。生长素的信号响应在很大程度上依赖于生长素的局部浓度，而植物中存在多种机制来调节生长素的浓度。因此，精确量化局部 IAA 浓度可以深入了解生长素的调节及其生物学作用。同时，生长素生物合成途径和基因尚未完全阐明，因此有必要在代谢物水平上分析生长素的产生，以便对生长素生物合成进行无偏研究。
我们开发了高通量方法来定量植物内源性 IAA 及其生物合成前体，包括吲哚、色氨酸、吲哚-3-丙酮酸（IPyA）和吲哚-3-丁酸（IBA）。该方案首先用加入稳定标记内标物的植物组织匀浆，然后使用固相萃取（SPE）小柱进行分析物纯化，最后通过气相色谱-质谱联用仪（GC-MS/MS）进行衍生化产物分析，以确定分析物的精确丰度。该测定法所需的植物组织量很小（通常为 2-10mg 鲜重），并且 SPE 小柱的使用简单方便，可在合理的时间内制备大量样品。
基于 SPE 小柱和 GC-MS/MS 的方法可实现从小量植物组织样本中高通量且准确地定量 IAA 及其生物合成前体。该方案可用于使用同位素稀释法测量这些内源性化合物，也可用于应用稳定同位素标记分析生长素生物合成和生物合成途径。该方法将有可能促进对生长素作用和调节的认识。
