Auxin measurements in plants are critical to understanding both auxin signaling and metabolic homeostasis. The most abundant natural auxin is indole-3-acetic acid (IAA). This protocol is for the precise, high-throughput determination of free IAA in plant tissue by isotope dilution analysis using gas chromatography-mass spectrometry (GC-MS). The steps described are as follows: harvesting of plant material; amino and polymethylmethacrylate solid-phase purification followed by derivatization with diazomethane (either manual or robotic); GC-MS analysis; and data analysis. [13 C 6]IAA is the standard used. The amount of tissue required is relatively small (25 mg of fresh weight) and one can process more than 500 samples per week using an automated system. To extract eight samples, this procedure takes ∼3 h, whether performed manually or robotically. For processing more than eight samples, robotic extraction becomes substantially more time efficient, saving at least 0.5 h per additional batch of eight samples.
Bibliographical noteFunding Information:
acknowleDGMents This work was supported by NSF grants DBI 0077769, MCB 0725149 and MCB 0725192 to J.D.C. and J.N., and in part by USDA-NRI Grant 2005-35318-16197 to J.D.C. and USDA-NRI 2005-03555 to J.N. We are grateful to A. Adepoju, A. Ahamed, J. Bernard, J. Calio, A. Davenport, C. Jensen, J. Ledoux, M. Lewandowski, Y. Mohammad, V. Patel and E. Sanni for conducting many trials of the extraction protocols. A. Culler has provided generous advice and discussions. We thank the many visitors to our laboratories over the last 5 years who tested our procedures as they were developed, especially M. Quint and R. Bottini. We thank other colleagues who have tested the method in their own laboratories, including J. Alonso and W. Coffeen. We also thank D. Bensen for the design and production of the custom metal bead dropper, insert plate and diazomethane generator.