New protocols for exploring the auxin metabolic network

Abstract

The phytohormone auxin plays a critical role in plant growth and development. Maintenance of auxin homeostasis involves a complex interaction between biosynthesis, formation and hydrolysis of conjugates, catabolism and transport. Despite significant progress in elucidating metabolic pathways of the primary bioactive auxin, indole-3-acetic acid (IAA), over the past few decades, key components such as intermediates and enzymes have not been fully characterized, and the dynamic regulation of IAA metabolism in response to environmental signals has not been completely revealed. Using a highly sensitive liquid chromatography-mass spectrometry (LC-MS) method, I identified auxin-amino acid conjugates in achenes of F. vesca as consisting of indole-3-acetylaspartate (IAasp) and indole-3-acetylglutamate (IAglu). In contrast to what has been proposed to occur in Arabidopsis, I determined that IAasp and IAglu are hydrolyzed by seedlings to provide a source of free IAA for growth. I also describe the methods for pathway analysis in Arabidopsis thaliana seedlings by monitoring incorporation of multiple stable isotopes from labeled precursors to IAA biosynthetic pathway intermediates coupled with chemical inhibitors application and high-performance LC-MS techniques. These methods were then adapted to survey Arabidopsis seedlings for their changing indolic profile. I describe in detail the Stable Isotope Labeled Kinetics (SILK) methods employing a rapid stable isotope labeling that allows for tracing the turnover rates of IAA pathway precursors and product concurrently with a time scale of seconds to minutes. By measuring the entire pathways over time and using different isotopic tracer techniques, I demonstrate that these methods offer more detailed information about this complex interacting network of IAA biosynthesis, and should prove to be useful for studying auxin metabolic network in vivo in a variety of plant tissues and under different environmental conditions.