Background: 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.Results: 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.Conclusions: 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.
|Original language||English (US)|
|State||Published - Aug 10 2012|
Bibliographical noteFunding Information:
This work was supported by the National Science Foundation (grants MCB-0725149, IOS-PGRP-0923960, IOS-PGRP-1238812 and MCB-1203438), the Minnesota Agricultural Experiment Station, and the Gordon and Margaret Bailey Endowment for Environmental Horticulture. We are grateful to L. Barkawi, W. Chen, and A. Culler for providing generous advice and discussions. We also thank staff from the Glygen Corp. for helpful technical support on the use of TopTips. We thank the many visitors to our lab who tested our methods with various plant species and tissue types and helped us improve these protocols.