The Auxin Biosynthesis Metabolic Network

Abstract

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. To trace the involvement of various biosynthetic routes to indole-3-acetic acid (IAA), I monitored label incorporation from three different stable isotope-labeled precursors ([13C6]anthranilate, [15N1]indole, and [13C3]serine) into a number of proposed biosynthesis intermediates as well as IAA under conditions that induce adventitious root formation in Arabidopsis hypocotyls. [13C3]Serine-derived 13C incorporation into IAA was nearly eliminated in seedlings treated with inhibitors targeting tryptophan aminotransferases and flavin monooxygenases of the YUCCA pathway (tryptophan→indole-3-pyruvate→IAA), suggesting this pathway is a significant contributor to the auxin pool in de-etiolating hypocotyls that can be effectively blocked using chemical inhibitors. Labeling treatment with both [13C6]anthranilate and [15N1]indole simultaneously resulted in higher label incorporation into IAA from [15N1]indole than from [13C6]anthranilate; however, this result was reversed in the proposed precursors that were monitored, with the majority of isotope label originating from [13C6]anthranilate. An even greater proportion of IAA became [15N1]-labeled compared to [13C6]-labeled in seedlings treated with YUCCA pathway inhibitors, suggesting a portion of IAA biosynthesis comes from an origin independent of the measured pool of Trp in these tissues. I also describe in detail the techniques I used for pathway analysis in Arabidopsis thaliana seedlings employing 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 in future studies exploring routes of IAA biosynthesis in vivo in a variety of plant tissues.