A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue

Lana S. Barkawi, Yuen Yee Tam, Julie A. Tillman, Ben Pederson, Jessica Calio, Hussein Al-Amier, Michael Emerick, Jennifer Normanly, Jerry D. Cohen

Research output: Contribution to journalArticlepeer-review

76 Scopus citations

Abstract

To investigate novel pathways involved in auxin biosynthesis, transport, metabolism, and response, we have developed a high-throughput screen for indole-3-acetic acid (IAA) levels. Historically, the quantitative analysis of IAA has been a cumbersome and time-consuming process that does not lend itself to the screening of large numbers of samples. The method described here can be performed with or without an automated liquid handler and involves purification solely by solid-phase extraction in a 96-well format, allowing the analysis of up to 96 samples per day. In preparation for quantitative analysis by selected ion monitoring-gas chromatography-mass spectrometry, the carboxylic acid moiety of IAA is derivatized by methylation. The derivatization of the IAA described here was also done in a 96-well format in which up to 96 samples can be methylated at once, minimizing the handling of the toxic reagent, diazomethane. To this end, we have designed a custom diazomethane generator that can safely withstand high flow and accommodate larger volumes. The method for IAA analysis is robust and accurate over a range of plant tissue weights and can be used to screen for and quantify other indolic auxins and compounds including indole-3-butyric acid, 4-chloro-indole-3-acetic acid, and indole-3-propionic acid.

Original languageEnglish (US)
Pages (from-to)177-188
Number of pages12
JournalAnalytical Biochemistry
Volume372
Issue number2
DOIs
StatePublished - Jan 15 2008

Keywords

  • 4-Chloro-indole-3-acetic acid
  • 4-Cl-IAA
  • Auxin
  • High-throughput methylation
  • High-throughput purification
  • IAA
  • IBA
  • IPA
  • Indole-3-acetic acid
  • Indole-3-butyric acid
  • Indole-3-propanoic acid
  • Organic acids
  • Plant hormones

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