Protocol: analytical methods for visualizing the indolic precursor network leading to auxin biosynthesis

The plant hormone auxin plays a central role in regulation of plant growth and response to environmental stimuli. Multiple pathways have been proposed for biosynthesis of indole-3-acetic acid (IAA), the primary auxin in a number of plant species. However, utilization of these different pathways under various environmental conditions and developmental time points remains largely unknown.

Here we describe techniques for pathway analysis in Arabidopsis thaliana seedlings using multiple stable isotope-labeled precursors and chemical inhibitors coupled with highly sensitive liquid chromatography-mass spectrometry (LC-MS) methods. These methods should prove to be useful to researchers studying routes of IAA biosynthesis in vivo in a variety of plant tissues.

Monitoring incorporation of stable isotopes from labeled precursors into proposed intermediates provides a method to trace pathway utilization and characterize new biosynthetic routes to auxin. These techniques can be aided by addition of chemical inhibitors to target specific steps or entire pathways of auxin synthesis. Conclusions: Here we describe techniques for pathway analysis in Arabidopsis thaliana seedlings using multiple stable isotope-labeled precursors and chemical inhibitors coupled with highly sensitive liquid chromatography-mass spectrometry (LC-MS) methods. These methods should prove to be useful to researchers studying routes of IAA biosynthesis in vivo in a variety of plant tissues.