A microtechnique was developed for the quantification of indole-3-acetic acid (IAA) in plant samples of one milligram fresh weight or less. The method permitted quantification of both free and conjugated IAA using a benchtop gas chromatograph-mass spectrometer. New methods for sample purification with high recovery at microscale levels, together with simple changes that result in enhanced sensitivity of the instrumentation, allowed for a significant reduction in the amount of plant material required for analysis. Single oat (Avena sativa L.) coleoptile tips could be studied with this method and were found to contain free and total IAA levels of 137 and 399 pg · mg-1 fresh weight, respectively. A single 5-d-old Arabidopsis thaliana (L.) Heynh. seeding was shown to contain 61 pg · mg-1 fresh weight free IAA and 7850 pg · mg-1 fresh weight of total IAA following basic hydrolysis. This microtechnique provides a way to accurately measure IAA levels in very small structures and individual seedlings, thus making it a valuable research tool for elucidating the role and distribution of auxin in relation to growth and development.
Bibliographical noteFunding Information:
We thank Dr. Kai-Hsien Chen (Hewlett Packard, Taiwan) for helpful suggestions which facilitated the development of this technique, Eric Schoettker (Hewlett Packard Co.) for help with repairs and modification of the mass spectrometer necessary for increased sensitivity and Drs. Jennifer Normanly (University of Massachusetts) and Martha Koester (Weyerhaeuser Co.) for helping with the development of this technique for use as a generally available method. We also thank Drs. Normanly and Koester as well as Drs. Harry J. Swartz (University of Maryland) and Janet P. Slovin (USDA-ARS Climate Stress Laboratory, Beltsville, Md., USA) for their helpful comments on the manuscript. Supported by U.S. Department of Energy grant DE-AI02-94-ER20153 and by the Maryland Agricultural Experiment Station.
- Arabidopsis (auxin analysis)
- Auxin analysis
- Avena (auxin analysis)
- Mass spectrometry
- Stable isotopes