SHORT COMMUNICATION Quantitative evaluation of IAA conjugate pools in Arabidopsis thaliana Peng Yu • Paula Lor • Jutta Ludwig-Mu ¨ ller • Adrian D. Hegeman • Jerry D. Cohen Received: 6 August 2014 / Accepted: 10 November 2014 / Published online: 25 November 2014 Ó Springer-Verlag Berlin Heidelberg 2014 Abstract Main conclusion This work has demonstrated that the major method of estimating the amount of unknown IAA conjugates—base hydrolysis—can be significantly complicated by chemical artifacts such as glucobrassi- cin or protein degradation. The concept of ‘bound auxin’ traces its origin back to more than 80 years ago and has driven research on the sources and forms of these plant hormones since. Indeed, analytical studies have demonstrated that the majority of cellular auxin is conjugated to simple sugars, cyclitols, glycans, amino acids, and other biomolecules. A number of studies have confirmed the enzymatic systems responsible for the synthesis and hydrolysis of a number of such conjugates in Arabidopsis thaliana and some of these compounds have been identified in situ. However, the amount of indole-3- acetic acid (IAA) released upon treating Arabidopsis tissue extracts with base, a commonly employed technique for estimating the amount of IAA conjugates, greatly exceeded the summation of all the IAA conjugates known individ- ually to be present in Arabidopsis. This discrepancy has remained as an unsolved question. In this study, however, we found that a significant portion of the IAA found after base treatment could be attributed to chemical conversions other than conjugate hydrolysis. Specifically, we showed that glucobrassicin conversion, previously thought to occur at insignificant levels, actually accounted for the majority of solvent soluble IAA released and that proteinaceous tryptophan degradation accounted for a large portion of solvent insoluble IAA. These studies clearly demonstrated the limits associated with using a harsh technique like base hydrolysis in determining IAA conjugates and support using more direct approaches such as mass spectrometry- based strategies for unambiguous characterizations of the total complement of IAA conjugates in new plant materials under study. Keywords Alkaline hydrolysis Á Auxin conjugates Á Glucobrassicin Á Mass Spectrometry Á Selective hydrolysis Abbreviations 4-Cl-IAA 4-Chloro-indole-3-acetic acid CV Column volumes DTT Dithiothreitol HEPES 2-[4-(2-Hydroxyethyl)piperazin-1- yl]ethanesulfonic acid IAA Indole-3-acetic acid P. Yu (&) Á P. Lor Á A. D. Hegeman Á J. D. Cohen (&) Department of Horticultural Science, Microbial and Plant Genomics Institute, University of Minnesota, 1970 Folwell Avenue, Saint Paul, MN 55108, USA e-mail: peng.yu@tum.de J. D. Cohen e-mail: cohen047@umn.edu P. Yu Á A. D. Hegeman Á J. D. Cohen Plant Biological Sciences Graduate Program, University of Minnesota, Saint Paul, USA Present Address: P. Yu Emil-Erlenmeyer-Forum 5, Chair of Proteomics and Bioanalytics, Technische Universita ¨t Mu ¨nchen, 85354 Freising, Germany J. Ludwig-Mu ¨ller Institut fu ¨r Botanik, Technische Universita ¨t Dresden, 01062 Dresden, Germany A. D. Hegeman Department of Plant Biology, University of Minnesota, Saint Paul, USA 123 Planta (2015) 241:539–548 DOI 10.1007/s00425-014-2206-z