Using targeted metabolomics to elucidate the indole auxin network in plants

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

Abstract

The plant hormone auxin plays important roles throughout the entire life span of a plant and facilitates its adaptation to a changing environment. Multiple metabolic pathways intersect to control the levels and flux through indole-3-acetic acid (IAA), the primary auxin in most plant species. Measurement of changes in these pathways represents an important objective to understanding core aspects of auxin signal regulation. Such studies have become approachable through the technologies encompassed by targeted metabolomics. By monitoring incorporation of stable isotopes from labeled precursors into proposed intermediates, it is possible to trace pathway utilization and characterize new biosynthetic routes to auxin. Chemical inhibitors that target specific steps or entire pathways related to auxin synthesis aid these techniques. Here we describe methods for obtaining stable isotope labeled pathway intermediates necessary for pathway analysis and quantification of compounds. We describe how to use isotope dilution with methods employing either gas chromatography or high performance liquid chromatography mass spectrometry techniques for sensitive analysis of IAA. Complete biosynthetic pathway analysis in seedlings using multiple stable isotope-labeled precursors and chemical inhibitors coupled with highly sensitive liquid chromatography-mass spectrometry methods are described that allow rapid measurement of isotopic flux into biochemical pools. These methods should prove to be useful to researchers studying aspects of the auxin metabolic network in vivo in a variety of plant tissues and during various environmental conditions.

Original languageEnglish (US)
Title of host publicationBiochemical Pathways and Environmental Responses in Plants
Subtitle of host publicationPart A
EditorsJoseph Jez
PublisherAcademic Press Inc.
Pages239-278
Number of pages40
Volume676
ISBN (Print)9780323955614
DOIs
StatePublished - Jan 2022

Publication series

NameMethods in Enzymology
Volume676
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
The work in the authors' laboratories was supported by NSF MCB 2225057, IOS-PGRP 2025297 and IOS-PGRP 1238812, by grants from USDA National Institute of Food and Agriculture, No. 2018-67011-28056, 2018-67013-27503 and 2019-51181-30025, Minnesota Department of Agriculture grant CON000000092336_2021 SCBG and by funds from the Minnesota Agricultural Experiment Station and the Gordon and Margaret Bailey Endowment for Environmental Horticulture. We thank Dr. Molly Tillmann, whose work contributed in important ways to the methods described. This paper is dedicated to the memory of Dr. Axel Ehmann whose early vision for quantitative mass spectrometry and his mentoring of the first author will be long remembered.

Publisher Copyright:
© 2022 Elsevier Inc.

Keywords

  • Auxin biosynthesis
  • Isotope dilution analysis
  • Mass spectrometry
  • Metabolic inhibitors
  • Pathway analysis
  • Stable isotope labeling
  • Metabolomics
  • Plant Growth Regulators/analysis
  • Gas Chromatography-Mass Spectrometry
  • Indoleacetic Acids/analysis
  • Indoles/metabolism
  • Plants/metabolism

PubMed: MeSH publication types

  • Journal Article
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, Non-U.S. Gov't

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